Sample-to-answer microfluidic cartridge

ABSTRACT

A microfluidic cartridge and methods for performing a diagnostic, molecular or biochemical assay thereon, where all dried and/or liquid reagents necessary for the assay are contained in the cartridge and the assay requires only the addition of sample. Pneumohydraulic features, chamber and diaphragm technologies are introduced for overcoming the problems of bubble interference and reagent washout during operation of a microfluidic cartridge. The cartridges are inserted into a host instrument for performance of an assay and the cartridge is supplied as a consumable.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit under 35 USC §119(e) to U.S.Provisional Patent Application No. 61/299,534 filed Jan. 29, 2010, whichapplication is incorporated herein by reference in its entirety.

BACKGROUND

1. Field

This disclosure is directed to microfluidic devices and methods fordiagnostic, molecular, and biochemical assays and, more particularly, tomicrofluidic technologies for dispensing and distributing fluid fromon-cartridge reagent reservoirs, for pumping, heating and mixing, andfor rehydrating dried reagents without bubble entrainment and withoutreagent washout.

2. Description of Related Art

Microfluidic devices have found increasing use as tools for diagnosticassays. The devices described by Wilding in U.S. Pat. No. 5,304,487consisted of “mesoscale” channels and chambers formed on reusablesilicon substrates which were infused with fluid reagents fromoff-cartridge syringe pumps. No consideration was given to on-cartridgefluid and reagent storage and delivery. However, practical commercialapplications have lead in the direction of “consumable”cartridges—disposable, single use “sample-to-answer” cartridges that areself-contained for all reagents needed for a particular assay or panelof assays. This is particularly true in the case of molecular biologicalassay applications, where contamination associated with sample carryoveror handling absolutely must be avoided.

On board reagents may include both liquid and dry reagent forms. Bothsuch reagent classes have been subject to certain problems inrealization of successful products. Here we address liquid handlingissues associated with initial wetout of the channels and chambers ofthe cartridge and with rehydration of dried reagents. During filling andoperation of a cartridge containing microfluidic channels and chambers,particularly those cartridges having a plastic body, liquid wetout isoften uneven, such that air pockets are not infrequently entrained inthe fluid column by the advancing meniscus against surfaces and incorners. During pumping and mixing of biological samples, foam andbubbles may form that negatively impact the assay performance of thedevice. Bubbles may arise due to uneven filling of channels or chamberscontaining dried reagents. Reagent rehydration, wetout and venting areinterlinked with the problem of bubble formation. The problem isexacerbated in more complex fluid networks such as described in U.S.Pat. Nos. 6,068,752 to Dubrow and 6,086,740 to Kennedy, for example, andin capillary flow-driven devices such as described by Buechler in USPatent Application No. 2005/0136552 or Wyzgol in US Patent ApplicationNo. 2004/024051, which have proved notoriously difficult in plastic bodydevices.

Bubbles may also arise during heating of a sample liquid due todegassing. It is well known that gas solubility is inversely related totemperature and that solutions which are heated readily becomesupersaturated. Also a source of bubbles by degassing is cavitation,where a fluid is sheared, such as during mechanical or ultrasonic mixingin microfluidic cavities.

Bubbles interfere with optical interrogation of liquids in microfluidic“cuvettes”. The path of light may be altered due to a lensing effectcreated by the curvature of the gas bubble surface and/or due to the gasbubble refracting the light. Bubbles may also interfere with biochemicalreactions by altering solute concentrations at bubble interfaces, bydenaturing protein structure, and by impacting bulk heating rate and thehomogeneity of temperature in a liquid. For example, in the PCRreaction, in which a thermostable polymerase is used to amplify copiesof a target nucleic acid, heating and cooling is uneven in the presenceof bubbles in the fluid, reducing the efficiency of the process andlimiting sensitivity. The presence of bubbles also reduces the volume offluid in the reaction chambers, and in assays which rely on detectinganalyte in volumes of 10-50 uL or less, the presence of a large trappedbubble in a reaction chamber can effectively kill the assay.

In reactions that rely on rate determination, bubbles can drasticallyinterfere with optical determination of slopes and with homogeneousrapid rehydration of dried reagents as is needed to start the reactionwith proper availability of substrates. A variety of dried reagents,such as a fluorescent probe, enzyme, buffer or control analyte, may beplaced within chambers of a microfluidic device and are needed forproper conduct of the assay. During wetout, entrapment of one or morebubbles may result in incomplete dissolution and mixing of the dryreagent and the sample, thereby impairing the reaction efficiency andreducing the sensitivity of the test.

Lei, in U.S. Pat. No. 6,637,463 proposes varying flow impedance inparallel channels through use of surface tension features and/orcross-sectional area so as to equalize pressure drops, and hence flow,through the multiple flow paths. In one instance, a plurality of exitchannels is used to drain fluid from a well so as to avoid formation ofrecirculating currents or fluid stagnation that would otherwise tend toinefficient washing of fluid and trapping of air bubbles. However, eachsuch feature must be designed by trial and error, and the designs arethus not robust or readily adapted for different assays. Becausemicroscopic variations in dimensions and surface chemistry are difficultto control in microfluidic circuit manufacture, the methods have notbeen proven a practical solution to the problem of equally dividing flowbetween parallel subcircuits within a microfluidic card. No descriptionof the use of diaphragms with features for improving wetout was offered.

Ulmanella (US Patent Application No. 2007/0280856) reported efforts tocontrol the meniscus of a fluid filling a microfluidic chamber byphysically modifying the bottom surface of the chamber, for example byinstalling an energy barrier to slow down or stop the leading edge ofthe meniscus as it crosses the floor of the chamber, or by use of aplurality of grooves or posts on the bottom surface, or by sculpting thedepth of the chamber so as to modulate capillary action, or by using asyringe pump, by centrifugation, or by application of a vacuum on theoutlet side of the chamber. None of these methods has proved a practicalsolution to the problem. Capillary action is highly unpredictable andtends to promote formation of air pockets and use of a syringe pump orapplication of vacuum, as commonly practiced in the prior art, tends toshear the fluid and drive fluid down the path of least resistance,further exacerbating the problem. For example, when two or moremicrofluidic channels branching from a single inlet are presented to afluid, such as is useful for splitting a sample or reagent betweenmultiple diagnostic assays pathways in parallel, the fluid may fill thepath most readily wetted and leave empty the path having higher fluidresistance. Very tiny differences in resistance between channels lead topreferential wetting of a single channel and no wetting of branchingparallel channels, a problem well known to those skilled in the art.

Ulmanella further addresses the effect of dried reagents in wetout ofmicrofluidic chambers and concludes that filling efficiency of chamberscontaining center-spotted dried reagent was less than 50%, chambershaving inlet side spotted reagent were wetted at 65% efficiency, but forchambers having outlet side spotted reagent, the filling efficiencywithout bubbles increased to 95%. However, positioning of reagent spotswith millimeter accuracy during manufacturing is neither a necessary nora satisfactory means of achieving wetout in the presence of driedreagent spots because it is preferential that the chamber be fullywetted before the reagent is rehydrated so that the concentration of thereagent is not diluted by washout into a downstream channel, as ishighly likely if the dry reagent is positioned at the downstream outletfrom the chamber!

It is further known that reduction in interfacial and surface tensionsin the microfluidic channels or chambers can be achieved, for example,by plasma treatment of the substrate(s) or incorporation of surfactantsto decrease hydrophobicity, and by applying a radius to channelintersections. These treatments are also known to improve wettability,but are not effective in eliminating mechanically entrained bubbles andbubbles resulting from thermal degassing, cavitation or stagnationzones. In fact, surfactants can increase the propensity of the gaseousphase to form stable bubbles and foams which can defeat performance ofthe assay by their persistence. Moreover, the modification of surfacesby processes such as plasma treatment are anticipated to be difficult tocontrol in manufacturing and may be impermanent, degrading progressivelyduring device storage. Therefore it is desirable and is an object ofthis invention to develop mechanical means and methods for reducing theformation and entrainment of bubbles during initial wetout of assaychannels, during rehydration of dry reagents, and for preventing orreducing accumulation and interference of bubbles during operation ofthe device.

BRIEF SUMMARY

Microfluidic cartridges of the invention, herein termed more generally“devices”, are generally formed of a flexible plastic body which housesfluidic channels and chambers patterned and fluidly intercommunicatingaccording to the needs of a diagnostic or biochemical assay to beperformed therein. The assay is conducted by reacting a sample with oneor more reagents in one or more steps, typically in one or more channelsor chambers of the device, for times and at temperatures effective informing a detectable product that indicates the presence or absence ofan analyte in the sample. The cartridges are typically consumables;i.e., they are used once and then discarded; and contain all reagentsneeded for one or more assays.

To perform an assay, a device of the invention is inserted into a hostinstrument which relies on optical detection (or other detection means),such as a spectrophotometer or fluorometer for the detection of achromogen or fluorophore indicative of the presence, absence, and/oramount of any target analytes of interest. In a preferred embodiment,optical windows in the device are interfaced with detection means in thehost instrument. However, the presence of one or more gas bubbles in anoptical window may impair the detection of the analyte. Bubbles may alsointerfere with the reactions required to form a detectable product, suchas for example an amplicon or other product of a biochemical ormolecular reaction, where a bubble may be responsible for uneven heatingof a reaction mixture, inadequate mixing, or incomplete or untimelyreconstitution of a dry reagent.

In use, a sample fluid is introduced into the inventive device, and thefluidly intercommunicating channels and chambers of the device are thenwetted with either a biological liquid sample alone, with liquidreagents, or with a mixture of a sample and one or more liquid reagents.The wettable, fluidly intercommunicating aspects of the device aretermed the “hydraulic works” of the device and comprise one or moremicrofluidic subcircuits having channels and chambers. Control of thehydraulics is effected through pneumatically actuated valves, pumps anddiaphragms superimposed as a separate, secondary network or manifold ofchambers and channels in the device and supplied by external sources ofpressurized air and vacuum. This secondary network is termed the“pneumatic works” of the device. Thus the device is composed of aprimary “hydraulic network” for conveying a liquid or liquids and asecondary “pneumatic network” for conveying a gas. The pneumatic networkprovides a) process control and b) positive and negative pressure fordriving the liquid or liquids through the hydraulic network, accordingto valve and pump logic of a host instrument with which the cartridge isinterfaced for performing an assay.

Sample handling and mixing of liquid reagents; including rehydration ofany dry reagents disposed within the hydraulic channels and chambers ofthe device, has been problematic in that bubbles readily becomeentrained in the fluid during wetting of the hydraulics. Thisparticularly occurs during initial wetout, where bubbles are engulfed bya meniscus advancing rapidly through the device, and subsequently suchas by cavitation or degassing associated with mixing and heating. Thepresent invention addresses this problem through one or more fluidhandling mechanisms and methods.

Inventive mechanisms, features and methods include pneumohydraulicdiaphragms characterized as:

1) an elastic, energy-storing pneumohydraulic diaphragm configured forpassively storing a liquid volume under a hydraulic pressure andreleasing the liquid volume during wetout of a downstream channel orchamber of the wettable microfluidic subcircuit;

2) a duplexedly layered pneumohydraulic diaphragm having a liquid centerfor storing and releasing a liquid reagent;

3) a pneumohydraulic diaphragm configured for eliminating headspace froma hydraulic chamber during wetout; or

4) a pair of pneumohydraulic diaphragms comprising a firstpneumohydraulic diaphragm interfacing a first hydraulic chamber withvalved inlet and a second pneumohydraulic diaphragm interfacing a secondhydraulic chamber with valved outlet, and an elevated directlyintercommunicating channel between the first and second hydraulicchambers, wherein the pair is configured for reciprocally exchangingfluid through the intercommunicating channel by applying opposingpressure differentials across the first and second pneumohydraulicdiaphragms; and

where the hydraulic chambers and diaphragms are configured forpreventing or reducing bubble entrainment or reagent washout duringwetout, fill, pumping or rehydration steps of an assay.

In accordance with various exemplary embodiments, one or more liquidreagents are disposed in sealed reservoirs on the device asmanufactured. Dry reagents are printed or “spotted” in channels orchambers and are rehydrated at the time of use. The liquid reagentsfunction as buffers, diluents, solvents, eluants, wash reagents, and asrehydrating reagents. In these capacities, the liquids are dispensed asrequired from their sealed reservoirs into the hydraulics of the deviceby pneumatic actuation.

In a preferred liquid reagent embodiment, a sealed liquid storagereservoir of the invention is structured as a two-layered diaphragm witha liquid center, the duplex diaphragm sealedly separating the pneumaticsworks and the hydraulic works of the device. The duplex diaphragm iscomposed of two impermeable film layers separated by a liquid center andcrimped or fused around the edges and sealed in the device so that thediaphragm separates a hydraulic chamber and a pneumatic chamber. Theupper layer, which faces the pneumatics works of the device, is formedof a film having a composition for resisting puncture and the lowerlayer, which faces the hydraulic works of the device, is composed of afilm having a composition that is more susceptible to puncture.Pressurizing the pneumatic side of the diaphragm forces theliquid-filled reservoir against a sharp or “barb” disposed in a fluidreceiving basin and punctures the lower layer, but not the upper layer.Following rupture, liquid then flows into the hydraulic chamber and fromthere into the microfluidic wettable channels of the device. By applyingpressure on the pneumatic side of the diaphragm, one or more volumes ofreagent can be forced under pressure into the hydraulic works, and byreversing pressure, the fluid can be cause to reflux.

In this aspect, an inventive assay cartridge is characterized as havingtherein:

-   -   a) a duplexedly layered diaphragm sealedly separating a        pneumatic chamber of a pneumatic works and a hydraulic chamber        of a hydraulic works, the duplexedly layered diaphragm having a        first side facing the pneumatic works and a second side facing        the hydraulic works, a first layer forming the first side        thereof, and a second layer forming the second side thereof, the        first and second layers enclosing therebetween a liquid volume        as a liquid center;    -   b) a fluid outlet for receiving and conveying the liquid volume        to the downstream microfluidic subcircuit; and    -   c) a sharp or “barb” disposed in the hydraulic chamber, the        sharp for selectively rupturing the second layer and for        releasing the liquid volume into the hydraulic works when the        duplexedly layered diaphragm is piercingly urged into contact        with sharp by application of a pressure differential across the        diaphragm.

Surprisingly, the liquid may be released from the on-board reagentreservoir in a series of smaller liquid volumes by the action of serialpulses of pneumatic pressure applied to the first layer of thediaphragm, which remains intact.

Optionally the first layer of the duplexedly layered diaphragm is arupture-resistant layer and the second layer is a rupture-sensitivelayer. The liquid center may contain a liquid reactant, a buffer, arehydrating fluid, a solvent, or a diluent. On-board storage of liquidis useful for, for example, rehydrating a dry reagent disposed in adownstream chamber or channel, for rinsing a solid phase, for eluting atarget analyte or analytes from a solid phase substrate, for making adilution, for making a chromatographic separation, for actuating orstopping a reaction, or for detecting the target analyte or analytes,and minimizes the possibility of carry-over contamination. Optionallythe liquid volume is degassed and the duplexedly layered diaphragm isgas impervious. Advantageously, any entrained bubbles are likely to beresorbed in degassed liquids, and degassed liquids are not susceptibleto degassing on heating, such as is useful for thermocycling in PCR.

While the devices are generally planar, they may be mounted in the hostinstrument in a canted position (i.e. angularly with respect to a groundplane), typically at about 15 degrees from flat and are vented at adownstream aspect of each microfluidic subcircuit. As a liquid sample orreagent is introduced upstream into the hydraulic subcircuitry, air isdisplaced downstream and is vented. The liquid sample and reagentsprogressively fill and move through the device. By canting the card atan angle of 10 to 35 degrees, air in the device during priming (termedhere “wetout”) is found to be more readily displaced from the hydraulicworks. By careful management of the advancing meniscus during initialfill of the canted card, the problem of bubble entrainment, particularlyduring fill, is substantially reduced or prevented.

Thus optionally, the hydraulic works may be configured for operationwhen mounted at an angle of 10-35 degrees relative to the ground planeon a tilted stage of a host instrument and at least one hydraulicchamber is configured with an outlet and intercommunicating channelpositioned superiorly relative to that chamber for venting a gas ordischarging a bubble from the chamber.

In another aspect of the invention, entrainment of bubbles during wetoutis limited by a filling mechanism that involves passive relaxation of anelastically stretched or distended pneumohydraulic diaphragm. Thispassive mechanism was found to be superior to fill by capillarity and tofill by positive displacement pump action or vacuum. A liquid is firstforced under pressure into a specially designed manifold having a“pneumatic chamber” stacked on top of a “hydraulic chamber”; where thetwo chambers are separated by an elastic diaphragm stretched over theroof of the hydraulic chamber. Optionally, liquid may instead beaspirated into the lower chamber, but advantageously, the upperpneumatic chamber is vented and open to atmospheric pressure. Theposition of the two chambers, while termed “upper” and “lower” or “top”and “bottom” chambers for purposes of explanation, is relative, and isnot limiting on the operation of the device. As a liquid volume entersthe liquid-receiving chamber, the diaphragm is stretched to hold thevolume and resiliently stores the energy of deformation, a form ofpotential energy having a returning force and a spring constant.Diaphragm material and deformation conditions are chosen so that the“elastic limit” of the material is not exceeded. Then by opening a valveto a downstream channel or channels, the distendedly stretched diaphragmreturns to its relaxed state and fluid gently fills the downstream fluidstructures without entrainment of bubbles in the advancing meniscus.

This mechanism and method has proved startlingly advantageous where flowis split into multiple channels. By providing an upstream stagingmanifold with multiple liquid-receiving chambers having elasticdiaphragms, each with separately valved outlets that are opened insynchrony, the hydraulic pressure for initiating and sustaining liquidflow into multiple downstream fluidic subcircuits in parallel issegregated or “quantized” so that the flow into all channels isessentially equal and sufficient. Total pressure and volume perdownstream channel can be precisely calibrated by selection of thespring constant and the deformation of the elastic diaphragm member sothat the restoring flow of liquid into the downstream channel is thevolume required to fill the downstream channel to a desired mark; thedisplaced volume delivered by each diaphragm of the staging manifold isneither insufficient nor in excess for the fluidic operation ofsplitting flow equally among multiple parallel channels or subcircuits,a necessary fluidic operation in devices intended for multiple assays inparallel. This is a technological advance in the art. Any air downstreamis readily displaced by the advancing meniscus and is conveyed to adownstream vent by this means.

In this aspect, an inventive assay cartridge includes:

a) a staging manifold having a plurality of chambers, wherein eachchamber of the plurality of chambers is separated into a hydraulicchamber and a pneumatic chamber by an elastic, energy-storingpneumohydraulic diaphragm sealedly mounted therebetween, such that aliquid volume admitted through an inlet into each hydraulic chamber inseries or in parallel distends each energy-storing pneumohydraulicdiaphragm according to an isobaric pressure proportionate throughoutsaid staging manifold to the displacement volume thereof;

b) the inlet is valvedly closeable for equilibrating the hydraulicpressure throughout the staging manifold after filling is complete; and,

c) a plurality of vented downstream channels in parallel, wherein onethe channel of the plurality of channels is in fluidic communicationwith each hydraulic chamber of the staging manifold, each venteddownstream channel having a valve for closing during filling andpressurization and for opening during draining and depressurization,whereby the hydraulic pressure of the elastic, pneumohydraulic diaphragmin a distended state is passively converted to the work of advancing ameniscus during initial wetout of the plurality of vented downstreamchannels equally in parallel.

More generally, wetout or ‘priming’ is improved by harnessing themechanical properties an elastic, pneumohydraulic diaphragm in a fluidlydistended state to do the work of advancing a meniscus through awettable downstream microfluidic circuit fluidly connected thereto andthereby displacing any gas therein to a downstream vent without bubbleentrainment. This principle is particularly advantageous in equallysplitting a fluid into a plurality of downstream microfluidicsubcircuits in parallel. In this way, multiple assays may be conductedin parallel and a single sample may be split equally for parallel assayshaving separate downstream detection means. Surprisingly, the mechanicalproperties of the elastic diaphragm can be calibrated to fill one ormore downstream microfluidic subcircuits to a mark, as is useful inreconstituting a defined mass of a dried reagent in a defined volume,for example.

Microfluidic devices may typically also include at least one driedreagent disposed within the downstream hydraulic network. These reagentsare typically spotted or printed during manufacture. During an assay,the dried reagents are rehydrated by sample or by contact with a liquidreagent dispensed as described above. Serendipitously, we have foundthat the passive liquid wetting mechanism and method described here isadvantageously suited to the rehydration of dry reagents withoutentrainment of bubbles, another technological advance in the art.

In a related embodiment, we have found that by providing pneumaticallyactuated diaphragms in downstream chambers where dried reagents arespotted, the diaphragms overlying those reagent spots can be pressurizedso as to a) temporarily seal the reagent zone (typically central to andon the floor of the chamber) from contact with bulk fluid during thechamber wetting process and b) remove or expel essentially all of theheadspace above the dried reagent. When deformed so as to fill thehydraulic chamber, the diaphragm is not fully sealed around theperiphery of the chamber. Liquid entering the chamber around thediaphragm is shunted around the lower edges of the chamber and readilydisplaces any residual air, which is vented from the hydraulics duringfilling. By relaxing or by reversing the pressure differential acrossthe diaphragm, additional fluid is readily aspirated into the chamberwithout the formation or entrapment of gas bubbles. Reagents arerehydrated only after the downstream outlet of the chamber is valvedlyclosed, thereby reducing reagent losses to washout. The reduced deadvolume of the dry reagent chambers is thus turned to advantage. Happily,in this way, dry reagent spots can be precisely reconstituted with adesired volume of rehydrating reagent or sample, ensuring that thebiological activity of the reagent is quantitatively correct for theassay conditions, a useful refinement in art.

Thus the invention also may feature at least one microfluidic subcircuithaving a downstream reaction chamber with upstream inlet and downstreamvent, the downstream reaction chamber containing a dried reagent spot orspots, further characterized in that the pneumohydraulic diaphragm isconfigured to operate with a first position wherein the diaphragm isdistended against the floor of the chamber so as to displace headspaceair and form a protective temporary tent around and over the reagentspot or spots during wetout, and a second position wherein the diaphragmis relaxedly positioned or aspirated against the roof of the chamber soas to fill the chamber with the liquid volume and uncover and dissolvethe reagent spot at full strength without bubble entrainment or reagentwashout. The dried reagent spot may be a buffer, an enzyme, a co-enzyme,a co-factor, a polymerase, a primer, a molecular beacon, a probe, afluorophore, a dehydrogenase, an oxidase, a reactant, a chromogen, asubstrate, an antibody, an antigen, or a control.

Also claimed is a method for wetting a microfluidic cartridge whilelimiting bubble entrainment therein, which comprises:

a) pumping a liquid volume through an inlet and into a plurality ofhydraulic chambers forming a staging manifold of a microfluidic card sothat an elastic pneumohydraulic diaphragm overlying the liquid volume ineach said hydraulic chamber is stretchedly distended, therebyisobarically pressurizing the liquid volume in the plurality ofhydraulic chambers;

b) valvedly opening an outlet from each of the hydraulic chambers of thestaging manifold, each outlet with fluidic connection to a venteddownstream microfluidic subcircuit; and

c) splitting the liquid volume substantially in equal measure into eachsaid wettable downstream microfluidic subcircuit by passive relaxing thedistended elastic diaphragm—without bubble entrainment.

Wetting a microfluidic device by passive relaxation of an elasticdiaphragm is readily distinguished from wetting by capillary action orby active pumping, and has proven surprisingly advantageous inovercoming difficulties with bubble entrainment as are known in the art.

Also claimed is a method for wetting a microfluidic cartridge whichcontains dried reagent spots, while limiting bubble entrainment therein,which comprises:

a) pumping a liquid volume through an inlet and into a plurality ofhydraulic chambers of a microfluidic card so that an elasticpneumohydraulic diaphragm overlying the liquid volume in each thehydraulic chamber is distended, thereby isobarically pressurizing theliquid volume;

b) pressurizing a second diaphragm in a plurality of downstream reactionchambers, each downstream reaction chamber containing a dried reagentspot, the second diaphragm forming a protective temporary tent forsealing around and over the reagent spot and for displacing headspaceair from the downstream reaction chamber;

c) valvedly opening an outlet from each the hydraulic chamber, each theoutlet with fluidic connection to one of the plurality of downstreamreaction chambers;

d) wetting the downstream reaction chamber around the temporary tent anddisplacing any residual air from the reaction chamber by allowing thedistended elastic pneumohydraulic diaphragm to relax, the liquid volumeforming an advancing meniscus;

e) optionally closing a valve downstream from the downstream reactionchamber;

f) lifting the temporary tent and conveying a remaining part of theliquid volume into each reaction chamber, thereby dissolving the reagentspot at full strength without bubble entrainment or reagent washout. Thetemporary tent is lifted by relaxing or by reversing the pressuredifferential across the second diaphragm member.

In another method, pairs of chambers with pneumohydraulic diaphragms maybe used to aid wetout and reagent dissolution for PCR, and forreciprocally pumping fluid when interconnected in series by a channel.By application of alternating positive and negative pneumatic pulses toa first diaphragm in a first chamber, a second diaphragm in a secondchamber is driven in synchrony. The second diaphragm may be an elasticdiaphragm that functions in accommodating and elastically storing thepulsed energy of the first diaphragm. Mixing is readily achieved byconveying a liquid volume back and forth between the two chambers. Byproviding each hydraulic chamber with a thin heat exchange film andsuitable contact heating elements, “two-zone” PCR is readily achieved.In an improved device, the intercommunicating channel between thechambers is contoured and elevatedly positioned so that bubbles aregravitationally urged to clear the chambers during initial wetout andpumping, and will trap any additional bubbles that form during heating.The intercommunicating channel is preferably configured and contoured tobe operated at a tilt of 10-35 degrees and is positioned on the highside of the paired chambers so as to reduce interference from bubbles.Fluid is cycled between a first chamber at a denaturing temperature of atarget nucleic acid and a second at an annealing temperature. Theplastic body of the device limits parasitic heat capacitance of thedevice during PCR. Nucleic acid amplification at rates of 8 seconds orless per thermal cycle is readily achieved.

For PCR, amplification reagents are provided with the device. Typicallythe first chamber contains a first reagent or reagents and the secondchamber contains a second reagent or reagents. Typically the reagentsare spotted in a centric or pericentric zone in each chamber. Duringinitial wetout, the diaphragms in the chambers are inflatedly distendedto press down on and cover the reagents so as to limit rehydration andany washout that would otherwise occur as the meniscus of therehydrating fluid or sample dissolves the spotted reagents and carriesthem downstream with the solvent front. After initial wetout, a suctionpressure may be applied to the diaphragm so as to aspirate a fluid intothe chamber and dissolve the reagents therein. Alternatively, anupstream chamber may be pressurized so as to hydraulically inflate thedownstream chamber and dissolve the reagents. Fluid direction of flowmay be reversed one or more times so at to improve mixing andrehydration.

Thus the invention may also include a cartridge for use with a hostinstrument having thermal interfaces for “two-zone thermocycfing” and apneumatic interface with pneumatic means for driving and controlling aPCR amplification. The device works by reciprocating pneumohydraulicaction of paired diaphragms in two interconnected hydraulic chambers soas to cyclically denature and anneal a target nucleic acid, thecartridge advantageously having one or more wettability features of theinvention for improving wetout of the chambers with liquid withoutentrainment of bubbles. The device is also advantageous for dissolvingreagents in a fixed volume without washout losses during wetout,ensuring that primers, buffers and other reagents are at a fixedstrength when reconstituted.

Thus the various aspects of the invention offer novel utility inoperation of microfluidic cartridges for diagnostic and biochemicalassays and are found to be advantageous as mechanisms and methods forlimitation of the bubble interferences that have been a longstandingsource of problems with these devices.

In the following description, certain aspects and embodiments of theinvention will become evident. It should be understood that theseaspects and embodiments are merely exemplary and explanatory and are notrestrictive of the invention. Other features and advantages will becomeapparent from the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show two perspective views of a disposable, single-use,sample-to-answer microfluidic cartridge of the invention, the cartridgecontaining all reagents for an assay and requiring only introduction ofa biological sample.

FIG. 2 demonstrates insertion of the assay cartridge in a hostinstrument for performance of an assay thereon.

FIG. 3A is a detailed view of the cartridge as inserted in a mechanismof a host instrument. The mechanism includes a heating manifold shown inFIG. 3B and a pneumatic control interface.

FIG. 4 is an exploded view of disposable microfluidic cartridge withliquid center foil diaphragm packs carrying liquid reagents.

FIG. 5A is a perspective view of a microfluidic circuit for extractionof a nucleic acid target from a biosample; FIG. 5B is a schematic of theextraction process.

FIGS. 6A-G provide views of a reagent reservoir formed of a bilayeredduplex diaphragm with liquid reagent center and a sharp or “barb” forpuncturing and releasing the liquid into the hydraulic works of themicrofluidic device.

FIGS. 7A and 7B show a microfluidic cartridge canted with a tilt asmounted in a host instrument.

FIGS. 8A and 8B show a worms-eye view of a network of channels andchambers for performing PCR on a microfluidic cartridge; the positionsof dry reagents are also marked.

FIGS. 8C and 8D illustrate an alternative cartridge configuration inworm's-eye view.

FIGS. 9A-9L schematically depict a passive initial wetout mechanism withstaging manifold.

FIGS. 10A-10C depict the operation of a staging manifold wherebyreagents are rehydrated in preparation for PCR. The operational sequenceis continued in FIGS. 10D-G.

FIGS. 10D-10G illustrate a PCR amplification using dual chambers withreciprocating diaphragm action.

FIG. 11 describes the steps of a method for extracting nucleic acidsfrom a sample, where a bilayered duplex diaphragm with liquid center isused to dispense the reagents.

FIG. 12 describes the steps of a method for priming the microfluidicchannels of a hydraulic works with liquids dispensed from a bilayeredduplex diaphragm with liquid center.

FIG. 13 describes the steps of a method for rehydrating dry reagentswithout bubble entrainment.

FIGS. 14A and 14C are worm's eye views of a network of microfluidicchannels and chambers for reverse-transcriptase-mediate PCR. FIG. 14B isa detail view of an in-line chamber for production of cDNA.

FIGS. 14D and 14E illustrate an alternative configuration of a cartridgewith modified features.

FIG. 15 illustrates use of optical windows of a detection chamber of adevice of the invention for monitoring a fluorescent endpoint.

FIG. 16 depicts more detail of a two piece microfluidic card assemblyfor performing PCR and a pneumatic interface with gasket for interfacingthe cards with a compatible host instrument.

FIG. 17 is a plot showing a positive and negative fluorescence assay inthe detection chambers of the cartridge, including multiple scans of thesample while increasing the temperature of the reaction mix.

FIGS. 18A and 18B analyze the pooled data of FIG. 17. Scans of amolecular beacon-amplicon duplex demonstrate a FRET melting curvecapability of the cartridge when interfaced with a compatible hostinstrument.

FIG. 19 depicts FRET data for amplicons obtained with a device of theinvention when used in a host instrument compatible therewith.

DETAILED DESCRIPTION

Although the following detailed description contains specific detailsfor the purposes of illustration, one of skill in the art willappreciate that many variations and alterations to the following detailsare within the scope of the claimed invention. The following definitionsare set forth as an aid in explaining the invention as claimed.

Definitions

A “cartridge” is an analytical device designed for operation byinsertion into a host instrument. The host instrument supplies thepneumatic pressure, pulses, and detection means for performance of theassay. The cartridge contains hydraulic works and pneumatic works, andmay include embedded microfluidic “cards” with embedded microfluidicchannels and chambers. Sample and reagent liquids are conveyed in ahydraulic network of the cartridge or card; fluid flow is controlled anddriven by a pneumatic network that interfaces with the hydraulics atselected junctions, channels and chambers. Typically, the body of thecartridge or card is made of a flexible plastic and may be formed bylamination, molding or a combination thereof. Plastics may include, butare not limited to, polycarbonate, polyethylene terephthalate, cyclicpolyolefins, acrylates, methacrylates, polystyrene, graft and blockcopolymers, and composites thereof. A preferred cartridge is made fromrollstock and includes dry reagents printed thereon.

“Hydraulic works” of a device: includes the network or networks ofintercommunicating channels and chambers that are intended to be wettedby sample or liquid reagents in the course of an assay. The hydraulicnetworks are configured with microfluidic subcircuits for performing thesteps of an assay.

“Pneumatic works” of a device: includes the network or networks ofpneumatically actuated valves, pumps and diaphragms and interconnectingcircuitry and manifolds that are useful for powering and controlling thehydraulics of the device. The pneumatic works of the cartridge deviceinterface with positive and negative pressure sources on the hostinstrument and with valves, diaphragms, pumps and other pneumaticallyactuated elements that control and drive liquids in the hydraulicnetwork.

“Microfluidic works” of a device: include the hydraulic works formed ofa network or networks of internal channels and chambers wetted in thecourse of the assay and the pneumatic works formed of valve control andpump driving circuits powered by positive and negative pressure sourceson the host instrument.

The microfluidic works may be divided into microfluidic subcircuits,where each subcircuit comprises channels and chambers for performing aparticular function on a liquid sample or reagent. The microfluidicsubcircuits may be organized into serial subcircuits (such as forextraction, amplification and detection of a nucleic acid target ortargets) and parallel subcircuits and networks such as for simultaneousassay for multiple targets on a single sample by splitting the sample.

“Top”, “bottom”, “up”, “down”, “above”, “below”, “upward”, “downward”,“superior to”, “floor”, “roof”, and so forth are indications of relativeposition and not absolute position, unless reference is made to aspecific frame of reference, such as the “ground plane”, which is takenas orthogonal to an intersecting plumb line.

“Wetout” (“wet out”) refers to the initial hydration of a plasticsurface interior to the hydraulic works of a cartridge. Because ofinterfacial tension effects, initial wetout can involve overcoming asubstantial energy barrier and is a major factor in resistance tocapillary flow in these devices.

“Target analyte”: or “analyte of interest”, or “target molecule”, mayinclude a nucleic acid, a protein, an antigen, an antibody, acarbohydrate, a cell component, a lipid, a receptor ligand, a smallmolecule such as a drug, and so forth. Target nucleic acids includegenes, portions of genes, regulatory sequences of genes, mRNAs, rRNAs,tRNAs, siRNAs, cDNA and may be single stranded, double stranded ortriple stranded. Some nucleic acid targets have polymorphisms, singlenucleotide polymorphisms, deletions and alternate splice sequences, suchas allelic variants. Multiple target domains may exist in a singlemolecule, for example an immunogen may include multiple antigenicdeterminants. An antibody includes variable regions, constant regions,and the Fc region, which is of value in immobilizing antibodies. Targetanalytes are not generally provided with the cartridge as manufactured,but are contained in the liquid sample to be assayed; in contrast,“control analytes” are typically provided with the cartridge or areroutinely present in a sample of a particular type and are assayed inorder to ensure proper performance of the assay. Spiked samples may beused in certain quality control testing and for calibration, as is wellknown in the art.

“Means for Amplifying:” of which the grandfather technique is thepolymerase chain reaction (referred to as PCR) which is described indetail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, Ausubel etal. (Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. 1989), and in Innis et al., (“PCR Protocols”, AcademicPress, Inc., San Diego Calif., 1990). Polymerase chain reactionmethodologies require thermocycling and are well known in the art.Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of a targetsequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the template to form reaction products, excess primerswill bind to the template and to the reaction products and the processis repeated. By adding fluorescent intercalating agents, PCR productscan be detected in real time.

Other amplification protocols include LAMP (loop-mediated isothermalamplification of DNA) reverse transcription polymerase chain reaction(RT-PCR), ligase chain reaction (“LCR”), transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA), “Rolling Circle”, “RACE” and “one-sided PCR”,also termed “asymmetrical PCR” may also be used, having the advantagethat the strand complementary to a detectable probe is synthesized inexcess.

These various non-PCR amplification protocols have various advantages indiagnostic assays, but PCR remains the workhorse in the molecularbiology laboratory and in clinical diagnostics. Embodiments disclosedhere for microfluidic PCR should be considered representative andexemplary of a general class of microfluidic devices capable ofexecuting one or various amplification protocols.

Typically, nucleic acid amplification or extension involves mixing oneor more target nucleic acids which can have different sequences with a“master mix” containing the reaction components for performing theamplification reaction and subjecting this reaction mixture totemperature conditions that allow for the amplification of the targetnucleic acid. The reaction components in the master mix can include abuffer which regulates the pH of the reaction mixture, one or more ofthe natural nucleotides (corresponding to A, C, G, and T or U—oftenpresent in equal concentrations), that provide the energy andnucleosides necessary for the synthesis of nucleic acids, primers orprimer pairs that bind to the template in order to facilitate theinitiation of nucleic acid synthesis and a polymerase that adds thenucleotides to the complementary nucleic acid strand being synthesized.However, means for amplication also include the use of modified or“non-standard” or “non-natural” bases such as described in U.S. Pat. No.7,514,212 to Prudent and U.S. Pat. Nos. 7,517,651 and 7,541,147 toMarshall as an aid to detecting a nucleic acid target.

“Means for detection”: as used herein, refers to an apparatus fordisplaying an endpoint, ie. the result of an assay, which may bequalitative or quantitative, and may include a machine equipped with aspectrophotometer, fluorometer, luminometer, photomultiplier tube,photodiode, nephlometer, photon counter, voltmeter, ammeter, pH meter,capacitative sensor, radio-frequency transmitter, magnetoresistometer,or Hall-effect device. Magnifying lenses in the cover plate, opticalfilters, colored fluids and labelled probes may be used to improvedetection and interpretation of assay results. “Labels” or “tags”include, but not limited to, dyes such as chromophores and fluorophores;and chemoluminescence as is known in the prior art. QDots, such as CdSecoated with ZnS, decorated on magnetic beads, or amalgamations of QDotsand paramagnetic Fe₃O₄ microparticles, are a convenient method ofimproving the sensitivity of an assay of the present invention.Fluorescence quenching detection endpoints are also anticipated. Avariety of substrate and product chromophores associated withenzyme-linked immunoassays are also well known in the art and provide ameans for amplifying a detection signal so as to improve the sensitivityof the assay, for example “up-converting” fluorophores.

“Molecular beacon”: is a single stranded hairpin-shaped oligonucleotideprobe designed to report the presence of specific nucleic acids in asolution. A molecular beacon consists of four components; a stem,hairpin loop, end labelled fluorophore and opposite end-labelledquencher. When the hairpin-like beacon is not bound to a target, thefluorophore and quencher lie close together and fluorescence issuppressed. In the presence of a complementary target nucleotidesequence, the stem of the beacon opens to hybridize to the target. Thisseparates the fluorophore and quencher, allowing the fluorophore tofluoresce. Alternatively, molecular beacons also include fluorophoresthat emit in the proximity of an end-labelled donor.‘Wavelength-shifting Molecular Beacons’ incorporate an additionalharvester fluorophore enabling the fluorophore to emit more strongly.Current reviews of molecular beacons include Wang K et al, 2009,Molecular engineering of DNA:molecular beacons. Angew Chem Int Ed Engl,48(5):856-870; Cissell K A et al, 2009, Resonance energy transfermethods of RNA detection, Anal Bioanal Chem 393(1):125-35 and Li Y, etal, 2008, Molecular Beacons: an optimal multifunctional biologicalprobe, Biochem Biophys Res Comm 373(4):457-61. Recent advances includeCady N C, 2009, Quantum dot molecular beacons for DNA detection. MethodsMol Biol 554:367-79.

Fluorescence nucleic acid assays include amplification with taggedprimers and probe-based detection chemistries. Fluorescent products canbe assayed at the end of the assay, or by measuring the amount ofamplified product in real time. While not limiting, TaqMan Probe(Applied Biosystems) which relies on displacement andpolymerase-mediated hydrolysis of a 5′ reporter dye with 3′ quencherconstruct, FRET hybridization probes, dual oligo FRET-based probes(Roche), minor groove binder-conjugated hybridization probes (MGBprobes, Applied Biosystems), Eclipse probes, Locked NA Probes(Ddqon/Roche), Amplifluor primer chemistries, Scorpions primerchemistries, LUX primers, Qzyme primers, RT-PCR, among others, are allsuitable in the present invention. Fluorescent probes includeintercalating probes, such as Syber Green® (Molecular Probes), ethidiumbromide, or thiazole orange, FRET probes, TaqMan® probes (RocheMolecular Systems), molecular beacon probes, Black Hole Quencher™(Biosearch Technologies), MGB-Eclipse® probes (Nanogen), Scorpions™ (DxSLtd) probes, LUX™ primer-probes (Invitrogen), Sunrise™ probes (Oncor),MGB-Pleiades (Nanogen), and so forth. Recent advances in probetechnologies are reviewed by Lukhtanov E A et al, 2007, Novel DNA probeswith low background and high hybridization-triggered fluorescence, NuclAcids Res 35:e30, for example. Reverse transcriptase is used to analyzeRNA targets and requires a separate step to form cDNA. Recent advancesinclude Krasnoperov L N et al. [2010. Luminescent probes forultrasensitive detection of nucleic acids. Bioconjug Chem 2010 Jan. 19epub].

In addition to chemical dyes, probes include green fluorescent proteins,quantum dots, and nanodots, all of which are fluorescent. Molecules suchas nucleic acids and antibodies, and other molecules having affinity foran assay target, may be tagged with a fluorophore to form a probe usefulin fluorescent assays of the invention.

“FRET” (Fluorescence Resonance Energy Transfer)—is a fluorescencetechnique that enables investigation of molecular interactions. Itdepends on the transfer of energy from one fluorophore to anotherfluorophore (ie. a donor and a quencher) when the two molecules are inclose proximity such a when hybridized. Recent advances include CarmonaA K et al, 2009, The use of fluorescence resonance energy transfer(FRET) peptides for measurement of clinically important proteolyticenzymes, Ann Acad Bras Cienc 81(3):381-92.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”. Referencethroughout this specification to “one embodiment”, “an embodiment”, “oneaspect”, or “an aspect” means that a particular feature, structure orcharacteristic described in connection with the embodiment or aspect maybe included one embodiment but not necessarily all embodiments of theinvention. Furthermore, the features, structures, or characteristics ofthe invention disclosed here may be combined in any suitable manner inone or more embodiments. “Conventional” is a term designating that whichis known in the prior art to which this invention relates. “About” and“generally” are broadening expressions of inexactitude, describing acondition of being “more or less”, “approximately”, or “almost” in thesense of “just about”, where variation would be insignificant, obvious,or of equivalent utility or function, and further indicating theexistence of obvious minor exceptions to a norm, rule or limit.

Description Of The Drawings

Turning to the figures, FIGS. 1A and 1B show two perspective views of adisposable, single-use, sample-to-answer microfluidic cartridge of theinvention, the cartridge containing all reagents for an assay andrequiring only introduction of a biological sample. In thisrepresentative embodiment, the cartridge 100 includes a protectivechassis or body 102 with coverplate 103 for convenience in handling. Thecoverplate includes and contains an inlet port 104 for addition ofsample. The projecting nose 105 of the cartridge is inserted into adocking bay of a host instrument (FIG. 2). The projecting nose of thecartridge body includes optical window cutout 101 that aligns with abackside mirror of the docking bay for reflective transillumination andfluorescence detection, while not limited thereto, of a target analytewhen inserted into the host instrument. Also on the underside of thecartridge is a thermal interface 110 for heating zones of themicrofluidic cartridge and a disposable gasket 111 for sealedly seatingthe cartridge to a pneumatic control interface of the host instrument inthe docking bay. The cartridge body may include microfluidic cards asshown in FIG. 16; however microfluidic works may optionally be integralto the cartridge body.

FIG. 2 demonstrates reversible insertion (double arrow) of the assaycartridge 100 in a docking bay 201 of a host instrument 200. Performanceof an assay is controlled with an operator interface generally as shown.Optical window 101 aligns with a detection apparatus inside the chassis202 of the host instrument.

FIG. 3A is a detailed view of the cartridge as inserted into a mechanismof a host instrument. An inclined mounting plate 300 is used to anglethe mechanism (and the cartridge) at a fixed angle theta (cf. FIG. 7B),which aids in venting air and entrainment of bubbles during initialwetout. The host instrument includes an optics assembly withtrack-mounted scanning detector head 303 and motorized clampingmechanism 304 for interfacing with optical window 101 of the cartridge.The optics assembly and docking bay are mounted as part of a floatingstage that is bolted to the inclined mounting plate but issuspension-mounted so that the cartridge may be clamped against thethermal control module 310 and pneumatics interface ports 330 shown inFIG. 3B. Further description of a host instrument, docking bay, andoptics package is provided in copending World Patent Appl. Publ. No. WO2010/088514, titled “PORTABLE HIGH GAIN FLUORESENCE DETECTION SYSTEM,”which is incorporated herein in full by reference.

A thermal control module 310 and pneumatic control interface 330 withten pneumatic ports are shown in more detail in FIG. 3B, which includesa partial view of inclined mounting plate 300. The underside of acartridge (which is sealed with a thin layer of a heat-conductivepolymer as a thermal interface) contacts the upper surfaces of first,second, third and fourth “zone” heating elements (311, 312, 313, 314). Afan 315 is provided for cooling. The top face of the first heatingelement is provided with a mirror face 320 and operates in conjunctionwith the optics of host instrument for transillumination and capturingreflected light and/or fluorescent emissions through the optical window101 of the cartridge when aligned in the docking bay.

FIG. 4 is an exploded view of a disposable microfluidic cartridge 100with on-board liquid reagents in frangible liquid reservoirs. Eachreagent reservoir is a bilayered duplex diaphragm pack carrying a liquidreagent. The cartridge chassis supports reagent reservoirs (421, 422,423, 424) in separate wells 426 within the housing. The cartridge asillustrated here is a cartridge designed for PCR and includes fourliquid reagents. Optical window cutout 101 on the anterior nose 105 ofthe cartridge chassis 102 is again shown. Also inside the chassis underthe coverplate 103 is an adsorbent pad 430 for sequestering liquidwastes generated in the assay. The cartridge 100 is disposable and issealed to prevent loss of biohazardous waste. The sample inlet 104 onthe coverlid 103 of the device is the sole externally accessible fluidport in the device. All reagents (including any dry reagents and anyliquids reagents or rehydrating fluids) are provided within thestructure of the device.

On the underside of the cartridge chassis, two “cards” containingmicrofluidic works are provided, an “outboard card” 410 and an “inboardcard” 400. These cards are built up of laminated and/or molded layers'and contain hydraulic and pneumatic networks designed for a PCR assay.They are generally flexible and made of plastics such as polyethyleneterephthalate and polycarbonate, although not limited thereto. Disk 409is a glass solid phase adsorbent used in the extraction of nucleic acidsfrom the sample. A seal patch 425 is needed to seal the hydraulic worksof the outboard card after installation of the solid phase disk 409.

The outboard card 410 contains a fluidic circuit that works inconjunction with liquid reagent reservoirs 421, 422, 423, 424 and solidphase adsorbent disk 409 to extract nucleic acids by the protocoloutlined in FIG. 5B. The inboard card 400 receives purified nucleicacids via the fluidic interface (overlapping tongues 411 a/411 b forforming a card junction) between the two cards 400 and 410 and conductsamplification and detection within the hydraulic network of microfluidicchannels embedded in the card. The inboard card includes thin surfacefilms that form a detection window 101 a sealing the top and bottom ofdetection chambers enclosed within the card body. These chambers containless than 50 uL of fluid and are heated by contact with the heatingblocks of FIG. 3B. Gasket 111 is provided for sealing the pneumaticcontrol interface to the undersurface of the inboard card at card tongue411 b, which connects to and extends the pneumatic distribution manifoldof the host instrument within the microfluidic device.

FIG. 5A is a perspective view of the outboard card 411, which interfaceswith the cartridge chassis and liquid reagent reservoirs for extractionof a nucleic acid target from a biosample; FIG. 5B is a schematic of theextraction process. In the extraction process, which is based on theBoom method (U.S. Pat. No. 5,234,809), the sample is first mixed with alysis buffer, consisting of a mixture of a chaotropic agent and adetergent, and contacted with solid phase adsorbent 409. Followingwashing with multiple aliquots of wash buffer, which are conveyed towaste, the adsorbed nucleic acids 501 are eluted with a dilute buffersolution and transferred (open arrow, to FIG. 7A) through a fluidicallycommunicating port system under tongue 411 a to a staging manifold onthe inboard card 400. The liquid contents of this staging manifold areused for nucleic acid amplification as described below. In each step ofthe extraction, a liquid reagent is required. Each liquid reagent isstored in a bilayer foil diaphragm with a liquid center and the liquidis released under control of a pneumatic actuator that impels thetwo-layer diaphragm against a sharp, which ruptures (only) the lowerlayer of the diaphragm and forces the liquid into the hydraulic works ofthe cards. This process is illustrated in FIGS. 6A-6G.

FIGS. 6A-6G provide various views of a reagent reservoir pouch 600formed of a bilayered (i.e. two-layered) diaphragm (layers 602, 603)with liquid reagent center 601 and a “sharp” 610 or “barb” disposedbelow the reservoir, the sharp tip pointing upwards against the lower ofthe two diaphragm layers 603 a/603 b, in a sealed internal chamber 615formed with well 426. The sharp member 610 is shaped for puncturing andreleasing the liquid contents into the hydraulic works of themicrofluidic cartridge or card.

FIG. 6A illustrates a fluid-filled pouch or reservoir consisting of twodiaphragm layers surrounding a liquid center. The two layers areillustrated in a cross-section through the pouch in FIGS. 6B and 6C.Layers 602 and 603 enclose liquid center 601. The two layers are sealedat the edges 604. Foil coated layers of polyester and other plasticswere used in forming the diaphragm layers 602, 603. Top layer 602 isgenerally tough, flexible and resists puncture. Contrastingly, bottomlayer 603 is designed to be punctured by sharp 610 and to release itscontents into the microfluidic works of the cartridge via reagent outletchannel 611 (FIG. 6D), shown here not to scale. FIG. 6B describes abiconvex reservoir with diaphragm layers 602 a and 603 a surroundingliquid center 601 a with sealed edge 604 a, FIG. 6C describes aplanoconvex reservoir with diaphragm layers 602 b and 603 b surroundingliquid center 601 b with sealed edge 604 b, each having particularadvantages in assembly and use.

In FIG. 6D the reagent reservoir is shown mounted as a duplex diaphragmenclosing a liquid center 601 in a reagent chamber 615 of the cartridgehousing. Lip seals 605 isolate the pneumatic works 606 from thehydraulic works 612. While not limited thereto, lip seals 605 may beformed by gluing with a UV-actuated adhesive or other sealing methodknown in the art. When pressurized by air through pneumatic control port607, the lower surface of the duplex diaphragm assembly (600) is pressedagainst sharp 610 so that the bottom film layer 603 is ruptured, but nottop film layer 602 (FIGS. 6B-6C). In this way, the mechanism becomes amicro-dimensioned pneumatic diaphragm-actuated liquid dispenser.Surprisingly, once the liquid center is pierced, serial pneumatic pulsesmay be used to force successive microliter volumes of liquid throughoutlet channel 611 and into the hydraulic works. The reagent outletchannel 611 is in fluidic communication with channels and chambers ofthe hydraulic network involved in assay reactions dependent on wetting,mixing, eluting and so forth. Plastic cover layers 616 and 617 seal thechamber 615.

FIGS. 6E-6G provide detailed views of the sharp member 610, which isdesigned so that puncture of lower film layer 602 is not self-sealingaround the contour of the sharp. FIG. 6E is a face elevation view; FIG.6F is a side elevation view, and FIG. 6G is a CAD-generated isometricview. While not limited to the precise form and detailed dimensionshown, the sharp is formed as a bisected cone 620 or frustrum of a conewith a barb tip 621, a planar first face 622 that is modified by themolded addition of a protruding convex 2^(nd) facet 623 and a recessedconcave 3d facet 624, which forms the mouth of outlet channel 611. Thedelicately molded concavity (concave 3d facet 624) in the projecting tipof the sharp, particularly in combination with the male convexity of the2^(nd) 623, confounds the tendency of the film layer to close therupture in diaphragm 603, thus ensuring operation as what is essentiallya pneumatically actuated “spigot” formed for piercing and draining theliquid centered diaphragm. The spigot remains open and fluid flowsfreely in response to controlled pneumatic pressure applied via port607. Pan 625 aids in draining the fluid of the reservoir into outletchannel 611.

After extensive experimentation, the piercing action of the sharp wasfound to be most advantageously effective when the barb tip 621 of thefrustrated cone was brought to a radius of from 0.004 to 0.0045 inches,and a preferred radius for this feature as determined to be 0.004 inches(four thousandths of an inch). Sharps outside the range where not foundto be as effective by comparison. A microfluidic cartridge of theinvention optionally may be characterized as having a sharp for piercinga reagent reservoir where the sharp is a frustrum section of a cone, thecone formed with a tip for selectively piercing a puncture sensitivelayer of a duplex diaphragm, the tip having a cutting point with radiusof 0.0040 to 0.0045 inches.

The frustrum section of the cone is provided with a planar first facet,a convex second facet formed on the planar first facet, and a concavethird facet formed on the concave second facet, the concave third facetforming a mouth of a fluid outlet descending therefrom for draining thereleased liquid into the hydraulic works.

In a preferred embodiment of the reagent reservoir with liquid center,the first layer of the duplexedly layered diaphragm is rupture resistantand the second layer, proximate to the sharp, is rupture sensitive. Thefirst layer may be a laminated polymer with outer nylon film configuredto be puncture resistant and the second layer may be a laminated polymerwith outer polyethylene terephthalate film configured to be puncturesusceptible. Suitable polymer layers may also contain a sandwichedmetallized layer, and are available for example from Technipaq Inc(Crystal Lake, Ill.), with a laminated polyethylene/metal/polymerbacking sandwich trilayer structure. An opposable polyethylene filmbetween the two diaphragm members of the fluid pouch is useful to permitheat sealing. UV-activated glues may be used to form a seal or gasketfor assembling the diaphragm in a cartridge housing.

FIGS. 7A and 7B show the inboard microfluidic card 400 canted with atilt as mounted in a host instrument. The card is inclined at about 15degrees (θ) on its side as detailed in FIG. 7B, which is a sectionalview through three detection chambers enclosed in the card. The tilt ofthe card is configured so air in the card is buoyantly directed to oneor more venting ports during wetout and fill, and any bubbles that doarise are trapped in upstream channels and chambers of the card and arelimited in entry into the heated zones and detection chambers of thecard. Fluid 501 from the nucleic acid elution operation of FIG. 5Benters the inboard card as shown and is routed into a network ofmicrofluidic channels and chambers described in the following figure. Atilt of 10 to 35 degrees has been found to be useful in reducinginterference by bubble entrainment and may be implemented for automatedassay systems by configuring the host instrument to accommodate a cantedstage whereupon a microfluidic card or cartridge is supported during theassay. A vibration assist may also be provided to further isolatebubbles from critical paths. These features also aid in removing airduring initial wetout, thus reducing the overall air available forbubble formation.

FIGS. 8A and 8B show a “worms-eye” view of a network 800 of channels andchambers for performing PCR as within a microfluidic card 400. Theillustration depicts the appearance of the internal wettable surfacesforming a microfluidic subcircuit, but depth of the channels andchambers is exaggerated for clarity. As shown in FIG. 8A, where threechannels a, b and c are depicted, eluate 501 (containing any nucleicacids of a sample) is ported into the card through via 801 and enters athree-chambered staging manifold 802′, the purpose of which is to splitthe fluid into three downstream channels equally and to gently andevenly urge the fluid into downstream chambers 804 and 805 whileavoiding entrainment of bubbles during initial wetout of internalplastic surfaces. Valves 811 are initially closed. The mechanism of FIG.8B depicts a single channel.

The splitting of a liquid volume 501 between multiple channels initiallywas found to be problematic because of uneven wetting, but is desirableso that multiple amplifications or assays can be performed in parallel.As reduced to practice, during the first stage of the filling process,liquid 501 enters three chambered manifold 802′ under pressure. Each ofchambers 802 a, 802 b, 802 c is bisected horizontally by an elasticdiaphragm (see FIGS. 9I-9L, 900) that segregates the fluid contents froman interfacing pneumatic chamber (i.e., the vented upper cavity in astack of two cavities separated by a diaphragm) and passively stretchesduring fill. During this step, pressure is equalized between themultiple channels. During the fill, air beneath the diaphragms exitsthrough vent 803, which contains as a sanitary feature a gas permeable,liquid impermeable filter membrane that seals when wetted. Continuedpressurization inflates the diaphragms in chambers 802, so that whenreleased by opening valves 811 (and all downstream valves thereto), thepressurized liquid flows evenly into the three (or more) parallelchannels as urged by a restorative spring force or pressure exerted bythe elastic diaphragm 900, which is distended during filling of chambers802. Because the restorative pressure can be precisely controlled andlimited, and is a function of the spring constant of the diaphragm, andbecause the displacement volume of the elastic diaphragms can beprecisely controlled, the extent of wetout or “priming” of thedownstream channels can be precisely calibrated in the manner ofvolumetrically filling a pipet, a clear advance in the art. Elastomericdiaphragms were achieved with polyurethane, polyvinylidene chloride,and/or polyester as diaphragm material. One such material is Saranex™(Dow Chemical), which is a polyvinylidene chloride extruded sheetsandwiched between polyolefin layers as a composite thin film. Othermaterials may be used.

Advantageously, the passively stretching diaphragms 900 (FIGS. 9A-9L) ofeach chamber 802 thus become an energy storing device for distributingfluid into one or more parallel downstream channels without entrainmentof bubbles. By knowing the downstream volume, the energy in thestretched diaphragms may be adjusted so that each parallel channel isfilled to a mark, as in a volumetric pipet, the fill volume generallyfalling short of the detection chambers 806 and final valve structure812 in each branch, but fully wetting chambers 804 and 805. Duringwetout, all downstream structures are cleared of air ahead of a steadilyadvancing meniscus via terminal vent 807, which may be operated undersanitary conditions by capping with a hydrophobic liquid impermeablegas-permeable membrane in the manner illustrated for vent 803, ifdesired. Because the flow of liquid during relaxation of the diaphragmsis not forced by pneumatic overpressure, does not depend on capillaryflow, and is finite, the advance of the meniscus is progressive andorderly, limiting entrainment of air pockets in its wake. This is atechnological advance in the art, permitting precise filling of paralleldownstream networks without entrainment of bubbles. The method isfacilitated by the tilt of the card and by removing corner radii (as aresometimes associated with localized increases in surface tension thatmay impede wetting) from junctions of channels and chambers.

In a further refinement of this method, chambers 804 and 805 are alsofitted with internal diaphragms. Unlike the passively flexing diaphragmof chamber 802, the pneumatic face of the diaphragms of chambers 804 and805 are not vented to atmosphere and can be driven by positive pneumaticpressure or negative pneumatic pressure supplied from a pressuremanifold, thus serving as pumps. During the fill cycle, the diaphragmsare “tented” or “inflated” downward to occupy volume of the lowerhydraulic chamber so to as to reduce or eliminate any dead volume of thechambers. Liquid seeping past these diaphragms on the outside bottomedges of the chambers fully wets the chambers and displaces any residualair. Then upon releasing the diaphragms after closing valve 812, liquidis aspirated from upstream to fill and make up the volume of thechamber.

In a further refinement of this method, dry reagents are placed inchambers 804 and 805, the nature of the dry reagents relating to thenature of the assay to be performed. The reagents are generally spottednear the center of the chamber. During initial wetout, the diaphragm isfully tented downward to occupy the volume of the lower hydraulicchamber so as to reduce the dead volume therein and protectively coversand protects the dry reagent spots from dissolution and washout as thechamber residual dead volume is wetted. After valve 812 is closed andthe chamber is flooded with liquid by reversing pressure differentialacross the diaphragm, the reagent dissolves rapidly and at fullstrength.

The positions of dry reagents are marked in FIG. 8B. As can be seen, dryreagents having specific functions are placed in designated chambers.Dry reagent spot 821 contains for example master mix and primers thatare advantageously mixed with and denatured in the presence of targettemplate. This chamber 804 is preferentially heated at a temperaturesufficient for the denaturing of template nucleic acid. Chamber 805contains for example dried polymerase 822 and is at a temperaturesuitable for annealing of primers and target and for initiation ofpolymerization. In the detection chamber 806, dry reagent spot 820contains probes such as, for example, “molecular beacons” orintercalation dyes which are used to detect amplicon produced in theamplification. Detection chamber 806 is bounded at a “top” and a“bottom” by thin film optical windows and is reflectivelytransilluminated for fluorometric detection of amplified target.Synergically, the bottom thin film layer is also effective in heattransfer from the mirror faced heating element shown in FIG. 3B, withwhich the card interfaces during the assay, and can thus be termed a“thermo-optical window”, such as is useful in assaying by thermalmelting curve as will be described below.

FIGS. 8C and 8D describe an alternative cartridge 830 for PCR. In thiscartridge, sample 501 entering the cartridge under pressure at sampleinlet 831 is split at trifurcation 833 and fills each of three chambers832 a, 832 b, 832 c, which are independently vented at hydrophobic vents834. Each chamber 832 contains an elastic pneumohydraulic diaphragm,which when stretched during fill exerts a pressure on the liquid volumecontained in the chamber. The chambers may be filled by injecting aseries of pressurized volumes from an upstream pump. Fluid flow into thethree branches of the distribution manifold is not necessarily splitequally, but volume and pressure in each chamber (832 a, 832 b, 832 c)become isobaric and equalized as the staging manifold equilibrates.During the fill process, downstream valves 835 are closed.

After pressurization of the staging manifold 836 is completed andequilibrated, valves 835 are opened so that the elastic diaphragm ofchambers 832 can relax while passively urging the liquid contents intoamplification chambers 837 and 838. During this process, the diaphragmelements of chambers 837 and 838 are inflated to occupy the lowerhydraulic chamber so that headspace is removed and any dried reagents inthe chambers are protected from being washed away by the advancingmeniscus. PCR amplification is performed as described for FIGS. 8A and8B. Downstream valve 839 is opened to convey any amplification productsthrough an antechamber 840 to a detection chamber 846 by pressurizingdiaphragms in both chambers 837 and 838 while valve 835 is closed. Anyair is flushed out of the system through terminal vent 847.Advantageously, dried probe 841 printed or spotted in the antechamber isdissolved and mixed with amplicon prior to injection into the detectionchamber, which improves the transparency of the thermo-optical windowbounding the detection chamber and reduces or prevents autofluorescenceof certain dyes useful as molecular beacons or FRET probes. By operatingthe card at a tilt angle θ as described in FIG. 7, air is advantageouslypurged to a vented port superiorly disposed on the detection chamber.

As can be seen in FIG. 8D, the inlet, outlet, and venting ports ofdetection chamber 846 and the amplification chambers 837 and 838 areasymmetrically placed. When the cartridge or card is canted on a tiltedstage of the host instrument (referencing FIGS. 3A and 7B),communicating ports (842, 843) between the amplification chambers and atthe terminal venting port (848) associated with the detection chamberare elevated relative to the chambers themselves and are contoured toovercome any surface tension effects of the geometry. Air in the systemis thus preferentially flushed from the system by the advancing liquidduring wetout, which fills the lower aspects of the chambers first, andany bubbles generated by heating-associated degassing of the liquidduring PCR are preferentially trapped between the amplification chambersso as to not interfere with heat transfer, and do not enter thedetection chamber.

In more methodological detail, FIGS. 9A-9L present a simplifiedchronology and schematic of the steps or stages of passive initialwetout with staging manifold. Cross-sectional and plan views are shownso that the progress of the advancing meniscus may be seen. In the firstview, FIG. 9A, the diaphragm 900 in the staging manifold chamber 802 isshown to be upwardly distended, turgid with a liquid reagent enteringfrom the left through open valve 910, and the pneumatic face of thediaphragm is vented at 905 to atmosphere. Downstream chamber 903 is dry,valve 911 is closed. The initial dry state of reagent spot 905 ismonitored in plan view in FIG. 9B on the right.

In FIG. 9C, both valves 910 and valve 911 are closed and valve 912 isopen for venting. Diaphragm 900 is pressurized and is tented down overreagent spot 905; the footprint of the diaphragm in contact with thebase of chamber 903 is illustrated by a dotted line 902 a in FIG. 9D.

In FIG. 9E, valve 910 remains closed and valves 911 and 912 are open. Asshown in plan view in FIG. 9F, an advancing meniscus begins to enterchamber 903.

In FIG. 9G, liquid continues to wet chamber 903 and fill any dead volumeon the periphery of the chamber around the collapsed roof formed by thediaphragm. This process continues as shown by snapshot in FIG. 9I. Theprogressive deflation of the passively stretched diaphragm 900 inchamber 802 is shown on the left in timelapse snapshots in FIGS. 9G, and9I. It can be seen in FIG. 9K that pressure applied across diaphragm 902can be reversed when valves 910 and 912 are closed so that liquid isaspirated from the staging manifold chamber 901 and fills chamber 903,dissolving dried reagent 905. Complete dissolution is shown figurativelyin FIG. 9L. During this process, air has been effectively displaced fromthe wetted areas, first by elimination of deadspace in chamber 903, thenby the progressive elimination of residual air by the relaxation ofdiaphragm 900, and finally by sealing the purged system and aspiratingthe contents of chamber 802 into chamber 903 to solubilize and to bemixed quantitatively as a reagent solution. The volume of liquid fillingdownstream chambers can be precisely controlled by configuring adisplacement volume of elastic diaphragm 900 and chamber 802; the rateof passive downstream fluid wetout is controlled by selecting a springconstant for the elastic diaphragm 900.

FIGS. 10A-10C demonstrate a further advantageous use of the aboveinventive mechanism for priming a PCR reaction, where a system havingtwo zones for thermal cycling of the nucleic acid substrate andpolymerase is demonstrated. All fluidic systems are contained in a cardor cartridge body 1000. Staging manifold chamber 1001 is vented toatmosphere and contains a passive elastic diaphragm 1002 capable ofstoring a pressurized liquid, which enters from the left through valve1003. As shown in FIG. 10B, this diaphragm 1002 becomes distended duringfluid entry and valve 1003 is then closed. Diaphragms 1011 and 1021 inchambers 1010 and 1020, respectively, are pressurized to form aprotective tent over dried reagent spots 1012 and 1022, and to displacedeadspace air from the chambers as shown in FIG. 10B. Downstream valves1023 and 1033 are open at this stage so that displaced air is ventedfrom the system via terminal vent 1034. In FIG. 10C, valve 1004 isopened and liquid enters the two chambers where PCR will occur, first inamounts sufficient for priming the chambers. This sequence is continuedin FIGS. 10D through G. Fluid is introduced in an amount sufficient tofill the denaturation hot chamber 1010 but no more; applying a vacuum todiaphragm 1011 aids this process. FIG. 10D shows that the “hot” or“denaturing” chamber 1010 is under vacuum and liquid has been aspiratedto fill the chamber. After any dry reagent 1012 is quantitativelydissolved and nucleic acid denaturation is sufficient, the liquidcontents of chamber 1010 are transferred to the second chamber 1020 at atemperature suitable for annealing of primers so thatpolymerase-mediated extension may begin upon dissolution of reagent spot1022. FIG. 10E shows liquid pumped from the hot chamber to the“annealing” chamber by reversing the pressure differential across thetwo diaphragms. This process is again reversed in FIG. 10F,demonstrating the reciprocating pumping action of the two diaphragms inforcing the liquid back and forth between the hot zone at 1010 (which iscontacted with heating block 313, FIG. 3B) and the annealing zone at1020 (which is contacted with heating block 312). This reciprocatingpneumohydraulic action is the basis of nucleic acid amplification bythermocycling in the apparatus. Finally, the fluid with any amplicons isejected into the detection chamber as shown in FIG. 10G. The detectionchamber as shown here is fitted with a pair of optical windows 1035.

In a more complex configuration, an additional temperature station andassociated thermal interface with thermal block 314 (FIG. 3B) is used,for example, for reverse transcriptase mediated synthesis of cDNA priorto a PCR-type amplification process. Thus additional chambers may beuseful and the geometry and configuration may be varied, mutatismutandi, by logical extensions of the teachings of the invention. Duringwetout, diaphragms in each chamber are used to reduce initial deadspacevolume. Subsequently, application of pressure differentials across thediaphragms are used to harness serial diaphragm assemblies and valveelements as pumps and mixing elements for hydraulic movement of fluidvolumes through the hydraulic works of the devices. A first embodimentof a reverse-transcriptase device is shown in FIGS. 14A-14C, and will bedescribed in more detail below.

FIGS. 11-13 summarize the steps of the methods described above. FIG. 11describes the steps of a method for extracting nucleic acids from asample, where a bilayered duplex diaphragm with liquid center is used todispense the reagents. After starting the host instrument, a liquidsample is placed in a microfluidic cartridge and the cartridge isinserted in the docking bay of the instrument. The host instrument readsa bar code on the microfluidic cartridge indicating the type of assay tobe run. The liquid sample is aspirated into a mixing chamber and celllysis buffer is dispensed and mixed with the liquid sample. To dispensethe lysis buffer a “liquid-centered diaphragm” is urged by pneumaticactuation against a sharp, rupturing the lower layer of the diaphragmand pumping the liquid into the card. The sample lysate is thencontacted with a solid phase nucleic acid adsorbent positioned in achamber in the card and the depleted sample lysate is directed toon-board waste. Ethanolic solution is dispensed from a secondliquid-centered diaphragm member and used to wash contaminants from thesolid phase adsorbent. The wash step may be repeated. The washes aresequestered to on-board waste. The solid phase matrix is briefly driedunder a stream of air to remove residual solvent. Elution buffer is thendispensed from a final liquid-centered diaphragm reservoir and contactedwith the solid phase matrix. The eluate with eluted nucleic acids 501 isthen transferred to a staging manifold for entry into a detectionsubcircuit. In the example provided here, a nucleic acid assay with PCRamplification is conducted on the eluate. Other nucleic acidamplification methods are known in the art and, as would be understoodfrom the teachings and drawings herein, may be practiced byreconfiguration of the various components of the devices of theinvention

FIG. 12 describes steps of a method for “priming” (i.e., wettinglyloading) channels and chambers of a hydraulic works with liquidsdispensed from a bilayered duplex diaphragm with liquid center 601 aspictured in FIG. 6D. After eluting a nucleic acid extract 501 with anelution buffer released by rupturing a reagent reservoir containing thebuffer, the fluid can be oscillated when contacting solid phaseabsorbent 409 (FIG. 4) so as to efficiently take up adsorbed nucleicacids. The eluate is then pumped under pressure into a staging chamberof a microfluidic card so that an elastic diaphragm which covers thechamber becomes distended and stores the potential energy. The stagingchamber inlet is sealed and pressure throughout the staging manifoldequalizes rapidly. A downstream valve to each channel is then opened.All downstream fluid channels and chambers are vented during thisoperation, which is useful to wet out or prime the downstream wettablesurfaces. Advantageously, as the elastic diaphragm relaxes, releasingits stored energy, the elasticity of the diaphragm gently but firmlyforces a liquid volume into the downstream channels and chambers equallyin parallel, the advancing meniscus displacing any residual air withoutbubble entrainment, an advance in the art. The liquid volume is splitinto branching parallel fluid pathways in this way.

FIG. 13 describes steps of a method for rehydrating dry reagents withoutbubble entrainment or reagent washout. During manufacture of a cartridgeof the invention, a dried reagent spot is printed in the center of areagent chamber. The reagent chamber is configured with an overlyingpressurizable diaphragm. A liquid sample is added and the cartridge isinserted into the docking bay of a host instrument, which suppliespressure and valve commands for operation of the cartridge. The sampleis first pumped under pressure into an unvented staging chamber, whichdistends an elastic energy-storing diaphragm covering the liquid in thestaging chamber. The aforementioned downstream reagent chamber is ventedand the pressurizable diaphragm therein is pressurized so as to form aprotective temporary seal around and over the dried reagent spot. Thedownstream valve of the staging chamber is then opened; the elasticdiaphragm relaxes and elastic energy of the diaphragm's recovery gentlyforces sample fluid into the downstream reagent chamber, displacing anyresidual air around the protective temporary seal. Finally, the pressuredifferential across the pressurizable diaphragm is reversed or relaxed,uncovering the reagent spot and aspirating a full volume of liquid intothe reagent chamber so that the reagent spot advantageously dissolves atfull strength in the sample fluid without bubble entrainment, an advancein the art.

FIGS. 14A and 14C are worm's eye views of a network of microfluidicchannels and chambers for reverse-transcriptase-mediated PCR. A devicehaving three parallel channels a, b, and c is shown. Sample 501 is splitbetween the channels so that three (or more) separate multiplex assaysmay be performed in parallel, for example. Unlike previously depictedembodiments, here a reagent 1220 is printed in a channel 1205 ratherthan in a diaphragm-actuated chamber. The passive wetting principlearticulated in FIG. 9, however, is retained: liquid is expelled into thechannel by the passive relaxation of an energy storing diaphragm thathad been primed by an upstream pump. This principle is effective inlimiting entrained air and in balancing fluid flow in parallel channelsbranching from a common staging manifold, where each channel providedwith a discrete passive diaphragm. Devices utilizing this passivelydriven wetting principle, as realized herein, are an advance in the art,overcoming deficiencies associated with both capillary-wetted andactively-wetted devices of the prior art.

In one embodiment, FIGS. 14A and 14C show a network 1200 of channels andchambers for performing rtPCR within another microfluidic card of theinvention. Depths of the channels and chambers in this “worms-eye” vieware exaggerated for clarity. Eluate 501 (containing any nucleic acids ofa sample) is ported into the card through via 1201 and enters a fluidlyinterconnected three-chambered staging manifold 1202, the purpose ofwhich is to split the fluid into three downstream fluid pathways equallyand to gently and evenly urge the fluid through downstream valves 1204,reagent channel 1205, valve 1206 and into chamber 1207 while avoidingentrainment of bubbles during initial wetout of internal plasticsurfaces. Valves 1204 are initially closed.

During the first stage of the filling process, liquid 501 enters thepoly-chambered manifold 1202 under pressure. Each chamber 1202 isbisected horizontally by an elastic diaphragm (see FIG. 9, 900) thatsegregates the fluid contents from a vented upper pneumatic cavity inthe chamber and passively stretches during fill. During the fill, airbeneath the diaphragms exits through vent 1203, which contains as asanitary feature a gas permeable, liquid impermeable filter membranethat seals when wetted and allows an increase in pressure, distendingthe diaphragms. Continued pressurization inflates the diaphragms inchambers 1202 with liquid, so that when released by opening valves 1204(and all downstream valves thereto), the pressurized liquid flows evenlyinto the three (or more) parallel downstream channels as urged by arestorative force exerted by the elastic diaphragms. The restorativepressure can be controlled and limited, and is a function of the springconstant of the diaphragm. The volumetric displacement of the elasticdiaphragms can be controlled, so that the extent of wetout (or“priming”) of the downstream channels is calibrated in the manner ofvolumetrically filling a pipet q.s. to a mark. The capacity to equallysplit a sample is advantageous in performing assays in parallel in amicrofluidic device and has been problematic when attempted by capillaryflow and by suction or positive displacement methods (such as a syringepump) because there is no assurance that flow in each of the channelswill progress at an equal rate. Surprisingly, using the principle ofwetout driven by passive relaxation of mated diaphragms in a stagingmanifold, this problem is advantageously solved for multiple parallelchannels.

Chambers 1207 and 1208 are fitted with internal diaphragms thatinterface between a hydraulic chamber and a pneumatic chamber. However,unlike the passively flexing diaphragm of chamber 1202, the pneumaticfaces of the diaphragms of chambers 1207 and 1208 are not vented toatmosphere and can be actively driven by positive pneumatic pressure ornegative pneumatic pressure supplied from an external source, thusserving as pumps. During the fill cycle, the diaphragms are fullydistended down into the hydraulic cavity to as to reduce or eliminateany dead volume of the chambers. Liquid seeping past these diaphragms onthe outside bottom edges of the chambers fully wets the chambers anddisplaces any residual air. Then upon releasing the diaphragms afterclosing valves 1209, liquid is aspirated under suction pressure fromupstream and fills the entire volume of hydraulic chamber 1207, airhaving been entirely flushed from the system.

In a variant, one of the pneumatic chambers is vented to atmosphere, andis slaved to the action of the unvented diaphragm. The two chambers areisolated from the remaining circuit elements by valves. When the activediaphragm is pulsed with positive pressure, liquid is forced to theadjoining chamber; when the active diaphragm is pulsed with negativepressure, liquid is aspirated from the adjoining chamber. Optionally,the passive diaphragm may be an elastic diaphragm.

In a further refinement of this method, dry reagents are placed inchambers 1207 and 1208 and in channel 1205. The reagents are generallyspotted on the floor of a hydraulic chamber or channel where the breadthof the passageway permits access by a printing head. The reagent 1220spotted in channel 1205 comprises a reverse transcriptase and nucleotidesubstrates in a suitable buffer. Typically a PCR master mix and suitableprimers are provided as reagent spot 1221 in chamber 1207. Spot 1222 isa dehydrated Taq reagent spot. Spot 1223 includes optional detectionreagents, such as a fluorescent probe. Multiple separate spots may beprinted using a roll-type or sheet-type process in each chamber orchannel.

RNA target in the eluate 501 is converted to cDNA by the action ofreverse transcriptase, generally at a temperature of 20 to 45° C. Thisaction is effected within channel 1205 in the elution buffer, and isdepicted in more detail in FIG. 14B, where valves 1204 and 1206 areseparated by a modified channel segment 1205 containing a dried reagentspot 1220. The reagent, for example a reverse transcriptase, isdissolved in sample transiting the specially modified channel segment.Substrates and any cofactors for full enzyme activity are also provided.

During initial wetout, diaphragms in chamber 1207 and 1208 are fullydistended down into the hydraulic chamber so as to reduce the deadvolume therein and the covering provided by the diaphragm protectivelyseals the underlying dry reagent spot or spots from prematuredissolution and washout during wetting. Vent 1211 is open to exhaust airthat is displaced by entry of the fluid, generally as a smoothlyadvancing meniscus. After valve 1209 is closed and the chamber 1207 isfilled with liquid by reversing pressure across the diaphragm (i.e.aspirating the liquid into the chamber), valve 1206 is also closed. Anyspotted reagent dissolves rapidly and at full strength, withoutdilution, essentially as described with respect to FIG. 8 for a directPCR process, where there was no need to form a cDNA from an RNA target.Reconfiguration of the device is thus flexible and may be adapted to avariety of molecular assay processes.

Chamber 1207 is preferentially heated at a temperature sufficient forthe denaturing of template nucleic acid. Chamber 1208 contains forexample dried polymerase 1222 and is at a temperature suitable forannealing of primers and target and for initiation of polymerization.Hot start of PCR is initiated for example by dissolution of a Taqpolymerase reagent spot 1222 in chamber 1208. Then, by alternatingpressure applied to the diaphragms of the two chambers 1207 and 1208,fluid may be moved back and forth from denaturing to annealingconditions by a reciprocating pneumohydraulic action of the diaphragms,and chain elongation and amplification has been found to be successfulin generating amplicons during this process. In the detection chamber1210, dry reagent spot 1223 contains probes such as, for example,“molecular beacons” which are used to detect any amplicon produced inthe amplification. As before, detection chamber 1210 is bounded on topand bottom by thin film optical windows and is reflectivelytransilluminated for fluorometric detection of amplified target.Synergically, the bottom thin film layer is also effective in heattransfer from the mirror faced heating element shown in FIG. 3B, withwhich the card interfaces during the assay, and can thus be termed a“thermo-optical window”, such as is useful in assaying or confirmingamplicon identification by thermal melting curves as will be describedbelow (FIGS. 17, 18A and 18B).

FIGS. 14D and 14E describe an alternative cartridge 1230 for PCR. Inthis cartridge, eluate 501 entering the cartridge under pressure atsample inlet 1231 is split at trifurcation 1233 and fills each of threechambers 1232 a, 1232 b, and 1232 c, which are independently vented athydrophobic vents 1234. Each chamber 1232 contains an elasticpneumohydraulic diaphragm, which when stretched or distended exerts apressure on the liquid volume contained in the chamber. If needed, thechambers may be filled by injecting a series of pressurized volumes froman upstream pump; fluid flow into the three branches of the distributionmanifold is not necessarily split equally, but volume and pressure ineach chamber (1232 a, 1232 b, 1232 c) become equalized as the stagingmanifold equilibrates. During the fill process, downstream valves 1235are closed.

After pressurization of the staging manifold 1236 is completed, valves1235 and 1237 are opened so that the elastic diaphragm of chambers 1232can relax while passively urging the liquid contents into reversetranscription channel 1238. Reverse transcription is conducted underbuffer, substrate and temperature conditions adapted for reversetranscriptase; buffer and any enhancers are generally supplied as adried reagent spot 1257 in chambers 1238. The sample is then urged intoamplification chambers 1247 and 1248. Each amplification chamber isfitted with a pneumohydraulic diaphragm. During this process, thediaphragm elements of chambers 1247 and 1248 are inflated underpneumatic pressure so that headspace is removed and any dried reagentsin the chambers are protected from being washed away by the advancingmeniscus by the inflated diaphragms, which are tented into the hydraulicchambers to cover the reagent spots. PCR amplification is performed oncDNAs made by reverse transcription as described for FIGS. 14A and 14C.Downstream valve 1249 is opened to convey any amplification productsthrough an antechamber 1250 to a detection chamber 1251 by pressurizingdiaphragms in both chambers 1247 and 1248 while valve 1237 is closed. Ateach stage, any air in the system is flushed out through terminal vent1252. Advantageously, dried probe 1253 printed or spotted in theantechamber 1250 is dissolved and mixed with amplicon prior to injectioninto the detection chamber, which improves the transparency of thethermo-optical window bounding the detection chamber and reduces orprevents autofluorescence of certain dyes useful as molecular beacons orFRET probes.

As can be seen in FIG. 14E, the inlet, outlet, and venting ports ofdetection chamber 1251 and the amplification chambers 1247 and 1248 areasymmetrically placed. When the device is canted on a tilted stage ofthe host instrument (referencing FIGS. 3A and 7B), communicating ports(1254, 1255) between the amplification chambers and at the terminalventing port (1256) associated with the detection chamber are elevatedrelative to the chambers themselves and are contoured to overcome anysurface tension effects of the geometry. Air in the system is thuspreferentially flushed from the system by the advancing liquid duringwetout, which fills the lower aspects of the chambers first, and anybubbles generated by heating-associated degassing of the liquid duringreciprocal pumping for PCR are preferentially trapped between theamplification chambers so as to not interfere with heat transfer, and donot enter the detection chamber.

Alternatively, reverse transcriptase cDNA and amplification may beperformed in one of the cartridges of FIG. 8. This is achieved byspotting reverse transcriptase (MMLV-RT, AMV-RT) and substrates in firstamplification chamber 804 and by first incubating at 40 to 50° C.Nucleic acids are extracted in the presence of an RNAase inhibitor.Suitable buffers for one-pot rtPCR are described in the literature andresult in what is essentially a pre-amplification of RNA targets, thusimproving sensitivity and the range of detectable molecular targets.Buffer systems for one-pot rtPCR are described for example by Young[Young et al. 1993. Detection of Hepatitis C virus RNA by a combinedreverse transcription-polymerase chain reaction assay. J Clin Microbiol31:882-86] and by others. Generally an RNAase inhibitor is added.

FIG. 15 illustrates use of optical windows of a detection chamber of adevice of the invention for monitoring a fluorescent endpoint. Anobjective lens 1520 is used to transilluminate a detection chamber 1500holding a liquid sample 1501. The detection chamber is bounded by anupper optical window 1502 and a lower optical window 1503. The chamberrests on a mirror face 320 of a heating block 311, the heating blockthus fulfilling dual functions of reflecting back the optical path forreflected light rays absorbed or emitted by chromogens or fluorophoresin the chamber and for modulating the temperature of detection chemistryin the fluid. This configuration has value for example in FRET detectionand for confirmation of detection of nucleic acid targets.

Photons emitted by a target molecule 1510 may be emitted in a cone thatis capture by the objective lens or may be reflected from mirror face320, thus forming a virtual image 1511 of the target molecule, and againare captured by the objective lens, increasing sensitivity. Thedetection chamber is thus mirrored by a “virtual detection chamber”(dotted lines) in the body of the heating block 311. Advantageously,bubbles 1505 forming in the detection chamber are gravitationally urgedaway from the center of the chamber by the inclination angle theta atwhich the device is disposed in the docking bay within the hostinstrument (see FIG. 7B). Synergically, the mirror-smooth surface 320also improves heat transfer, and lower optical window 1503 also servesas a heat transfer film. The heat transfer film is advantageously verythin and is forced into thermal contact with heating block 311. Apreferred heat transfer film is described in U.S. Pat. Nos. 7,544,506and 7,648,835, which are coassigned, but may also include cyclicpolyolefin films of similar dimensions, for example. The assembly thusfunctions as a thermo-optical window, achieving improved heating andoptical interrogation of any amplicons or other detectable speciespresent.

FIG. 16 depicts a representative level of complexity of the microfluidicworks for performing PCR and a pneumatic interface with gasket forinterfacing the microfluidic works with a host instrument. Shown forpurposes of illustration are fluidic channels and chambers comprising ahydraulic works with microfluidic subcircuits and a pneumatic works foroperating a molecular detection assay, exemplary details of which havebeen described here. An outboard card 410 and an inboard card 400 arejoined at a common pneumatic junction which is sealed using a disposablegasket 405 during operation to the pneumatic control interface in orderto pneumatically control and drive the hydraulic workings of the cards.This subassembly 1600 is generally mounted in a cartridge chassiscontaining reagent reservoirs as described with reference to FIG. 4. Themicrofluidic works of the cards include the hydraulic works formed of anetwork or networks of internal channels and chambers that are wetted inthe course of the assay and the pneumatic works formed of valve controland pump driving circuits powered by positive and negative gas pressuresources on the host instrument. Diaphragm valves are pneumaticallyopened and closed to control steps of the assays. Larger diaphragmsdisposed at the interface between the hydraulic works and the pneumaticworks also serve as pneumohydraulic devices for moving fluids and alsofor converting kinetic motion of fluids into potential energy in theform of elastically distended diaphragm elements of a staging manifoldand/or passively driven pumps in the amplification chambers, forexample. In this figure, the outboard card 410 is responsible fornucleic acid extraction from a biological sample, and the inboard card400 is used for amplification and detection. Other combinations arereadily conceived within the scope and spirit of the invention, which isnot limited by the illustrative examples provided.

FIGS. 17, 18A and 18B are representative of the types of assay resultsobtained with the microfluidic cartridges of the present invention,while not limiting thereto. FIG. 17 shows scanning data collected for amolecular beacon hybridized to an amplicon. The scanning axis (x-axis ofplot) transects detection wells representing positive and negative testconditions respectively, and it can be seen that signal is limited tothe detection wells. In the figure, the sample is scanned repetitivelyas the temperature in the detection chamber is systematically varied.The scans are overlaid in the plot to illustrate the spatial fidelity ofthe optical scanning apparatus. Fluorescence scans for 35° C., 65° C.,70° C., 75° C. and 80° C. test conditions are marked. Test scans at 40,45, 50, 55, and 60° C., and the 85 and 90° C. plots were not welldifferentiated, as would be expected, and are not individually marked.It can be seen that fluorescent signal is a function of temperature.Fluorescence quenching in this example is observed to increase as thedouble stranded probe-target is melted, ie. signal is greatest at 35° C.and is essentially not present at 80° C. In FIG. 18A, the data isreplotted for signal versus temperature for the positive (2301, solidline) and negative (no target, 2302, dotted line) test conditions. InFIG. 18B, a first derivative is plotted, indicating a FRET melttemperature of about 70° C.

EXAMPLE I

By example, the apparatus of the invention is shown to be useful indiagnosis of infectious disease by detection of the nucleic acids of apathogen in a human sample such as feces. Of interest by way ofillustration were the rfb gene useful in genetically detectingEnterobacteriaceae-like O-antigen serotype and the stx₁ and stx₂ genes(for shigatoxins). These genes are found for example in Shigella,Salmonella, Campylobacter, and Escherichia coli serotypes of interest indiarrheal disease.

Negative fecal swabs were diluted in 2.5 mL of PBS and spiked withO157:H7 bacterial culture. Diluted samples 250 uL were loaded foranalysis onto a cartridge of the invention. These cartridges containedall required dried and liquid reagents for PCR and molecular beaconamplicon detection. After DNA extraction, target and primers weredenatured at 94 C for 2 minutes and then cycled for PCR amplification atabout 12 sec per thermal cycle. After loading, an instrument havingthermal, pneumatic and optical interfaces designed to be compatible withthe cartridge was used to run a multiplex nucleic acid assay on thesample. Bacteroides DNA was used as an internal positive control on theamplification; negative controls were also run and produce no falsepositives.

A FAM-labelled probe for bacteroides is detected by a first fluorescence(excitation 485 nm, emission 535 nm). A CAL fluor Red 610-labelled probe(excitation 590 nm, emission 610 nm) is used to detect the targetanalyte in this assay. Biplex amplification products were detected at ornear a minimum of 80 target copies per extract against an internalcontrol background estimated at 400,000 copies, indicating a high levelof sensitivity and specificity. Details of the optics are described inWorld Patent Application Publication No. PCT/US10/22581, titled PORTABLEHIGH GAIN FLUORESCENCE DETECTION SYSTEM, which is co-pending and isincorporated in full by reference for all purposes herein.

FRET curves for amplicons detected for stx2 (2411), stx1 (2412), and rfb(2413) genes in fecal samples in a device of the invention areillustrated in FIG. 19. Control data is not shown.

EXAMPLE II

Using on-board dry and liquid reagents, a blood sample may be processedand RNA associated with Measles virus detected in 30 minutes or less. Ina first step, cDNA is formed from the sample at an incubationtemperature of about 50 C in one of the devices shown in FIG. 14, andthe reverse transcriptase product is then subjected to PCR using twomicrofluidic chambers (1221, 1222) with dual temperature zone controlgenerally as described in U.S. Pat. Nos. 7,544,506, 7,648,835,7,763,453, and 7,763,453 which are co-assigned, and in pendingapplications titled, “Integrated Nucleic Acid Assays”, which areco-assigned and incorporated in full by reference for all purposesherein. Amplicon is then detected using a FAM fluorescence-taggedmolecular beacon directed at the amplified target. Optionally, a controlconsisting of a California Red-tagged RNAase P leukocyte exon sequence(which is generally characteristic of any genuine human blood sample)with multiplex amplification in a one-pot reaction mixture, is used tovalidate the assay.

Other examples illustrating various combinations of inventive elementsand features are readily demonstrated. Devices configured per theteachings of the invention may be used in molecular assays for a targetnucleic acid (either DNA or RNA) associated with, for example, aninfectious agent selected from a bacterium (including Acinetobacterbaumannii, Actinobacillus equuli, Bacillus anthracis, Brucellamelitensis, Brucella abortus, Bordatella pertussis, Bordatellabronchioseptica, Burkholderia pseudomallei, Corynebacterium diptheriae,Coxiella burnetii, Eikenella corrodens, Escherichia coli, Francisellatularensis, Francisella novicida, Fusobacterium necrophorum, Haemophilusinfluenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Kingelladenitrificans, Legionella pneumophila, Leishmania ssp, Listeriamonocytogenes, Moraxella catarrhalis, Mycobacterium tuberculosis,Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides,Pasteurella multocida, Proteus vulgaris, Proteus mirabilis, Pseudomonasaeruginosa, Pseudomonas putrefaciens, Pseudomonas cepacia, Salmonellatyphi, Shigella dysenteriae, Staphylococcus aureus, Streptococcuspyogenes, Streptococcus pneumoniae, Treponema pallidum, Yersinia pestis,or Vibrio cholera), a Rickettsial agent (including Chlamydia pneumoniae,Chlamydia trachomatis, Rickettsia prowazekii, or Rickettsia typhi), aviral agent (including Measles virus, HIV virus, Hepatitis C virus,Hepatitis B virus, Dengue Virus, Western Equine Encephalitis virus,Eastern Equine Encephalitis virus, Venezuelan Equine Encephalitis virus,Enteroviruses, Influenza virus, bird flu, Coronavirus, SARS Coronavirus,Polio virus, Adenovirus, Parainfluenza virus, Hanta virus, Rabies virus,Argentine Hemorrhagic Fever virus, Machupo virus, Sabia virus, Guanaritovirus, Congo-Crimean Hemorrhagic Fever virus, Lassa Hemorrhagic Fevervirus, Marburg virus, Ebola virus, Rift Valley Fever virus, KyasanurForest Disease virus, Omsk Hemorrhagic Fever, Yellow Fever virus,Smallpox virus, a retrovirus, Monkeypox virus, and foot and mouthdisease virus), a fungal agent (including Coccidiodes immitis, Candidaalbicans, Cryptococcus neoformans, Histoplasma capsulatum, Blastomycesdermatitidis, Sporotrhix schenki, or Aspergillus fumigates), a parasiticagent (including Plasmodium falciparum, Plasmodium vivax, Plasmodiumovale, Plasmodium malariae, Toxoplasma gondii, Plasmodium bergeri,Schistosoma mansoni, Schistosoma hematobium, Schistosoma japonicum,Entamoeba histolytica, Babesia, Toxoplasma gondii, Trypanosoma cruzi,Leishmania ssp, Trypanosoma brucei, Trichinella spiralis, Toxocaracanis, Necator americanus, Trichuris trichura, Enterobius vermicularis,Dipylidium caninum, Entamoeba histolytica, Dracunculus medinensis,Wuchereria bancrofti, Brugia maldi, Brugia timori, Strongyloidesstercoralis, Ascaris lumbricoides, Onchocerca volvulus, Naegleriafowleri, Clonorchis sinensis, Cryptosporidium parvum, Leishmania spp),or also a gene or a sequence including an antibiotic resistance gene, agene associated with virulence or toxigenicity, a molecular marker, asingle-nucleotide polymorphism, an insect gene, a bee disease agentgene, a plant gene, a plant disease agent, a molecular marker associatedwith a cell having a pathogenic or carcinogenic condition, amitochondrial nucleotide sequence, a plasmid sequence, a messenger RNA,a ribosomal RNA, or a panel of target nucleic acids, and the like, asmay be interesting or useful. And may be used in molecular diagnosis ofinfectious agents in a mammal or vertebrate, including livestock,veterinary and aquaculture applications broadly. And also diagnosticapplications in plants, animals or insects suffering more generally froma pathogenic condition, for example, infectious or otherwise.Immunological and biochemical assays employing cartridge devices havingthe features of the invention are also conceived and claimed fordiagnostic use.

While the above is a description of the preferred embodiments of thepresent invention, it is possible to use various alternatives,modifications, combinations, and equivalents. Therefore, the scope ofthe present invention should be determined not with reference to theabove description but should, instead, be determined with reference tothe appended claims, along with their full scope of equivalents. Theappended claims are not to be interpreted as includingmeans-plus-function limitations, unless such a limitation is explicitlyrecited in a given claim using the phrase “means for.”

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent literature and publications referred to in this specificationand/or cited in accompanying submissions, are incorporated herein byreference, in their entirety. Aspects of the embodiments may bemodified, if necessary to employ concepts of the various patents,applications and publications to provide yet further embodiments. Theseand other changes may be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the specifics of the disclosure.

What is claimed is:
 1. A microfluidic cartridge comprising a plastic,thermally insulative cartridge body enclosing: i) a hydraulic workscomprising an upstream sample inlet, one or more on-board liquid or dryreagents for an assay, and a wettable downstream microfluidic subcircuitcomprising a channel and chamber fluidly connected to said upstreaminlet and vented at a downstream vent; ii) a pneumatic works comprisingan inlet or inlets configured for receiving pneumatic pressure and apneumatic subcircuit comprising a channel and chamber configured forconveying said pneumatic pressure therefrom; iii) a staging manifoldhaving a plurality of chambers, wherein each said chamber of saidplurality of chambers is separated into a hydraulic chamber and apneumatic chamber by an elastic, energy-storing pneumohydraulicdiaphragm sealedly mounted therebetween, and configured such that aliquid volume admitted through an inlet into each said hydraulic chamberin series or in parallel distends each said energy-storingpneumohydraulic diaphragm according to an isobaric pressureproportionate to the displacement volume thereof, wherein said inlet isvalvedly closeable for equilibrating said hydraulic pressure throughoutsaid staging manifold after filling is complete; and; iv) a plurality ofvented downstream channels in parallel, wherein one said channel of saidplurality of channels is in fluidic communication with at least one ofsaid hydraulic chambers of said staging manifold, each said venteddownstream channel having a valve configured for closing during fillingand pressurization and for opening during draining and depressurization,whereby said hydraulic pressure of said elastic, pneumohydraulicdiaphragm in a distended state is passively converted to the work ofadvancing a meniscus during initial wetout of said plurality of venteddownstream channels in parallel, and wherein said hydraulic chambers anddiaphragms are configured for preventing or reducing bubble entrainmentor reagent washout during wetout, fill, pumping or rehydration steps ofan assay, and wherein said pneumatic chamber is vented to atmosphere. 2.The microfluidic cartridge of claim 1, wherein said hydraulic works isconfigured for operation when mounted at a tilt angle theta ranging from10 to 35 degrees relative to the ground plane on a tilted stage of ahost instrument and at least one hydraulic chamber is configured with anoutlet and intercommunicating channel positioned superiorly relative tosaid hydraulic chamber for venting a gas or discharging a bubble fromsaid hydraulic chamber.
 3. The microfluidic cartridge of claim 1,wherein said hydraulic works comprises a plurality of wettabledownstream microfluidic subcircuits with channels and chambers fluidlyconnected to said upstream inlet and vented at one or more downstreamvents, wherein said plurality of wettable downstream microfluidicsubcircuits are each configured for performing an assay in parallel, andeach said wettable downstream microfluidic subcircuit is provided with aseparate detection chamber.
 4. The microfluidic cartridge of claim 1,wherein the microfluidic cartridge is configured such that a hydraulicpressure and liquid volume of at least one said elastic, energy storingpneumohydraulic diaphragm in a distended state is passively converted toa work of advancing a meniscus in a wettable downstream microfluidicsubcircuit fluidly connected thereto by opening a downstream valve,thereby displacing any gas to said downstream vent.
 5. The microfluidiccartridge of claim 4, wherein said liquid volume and hydraulic pressureof said at least one elastic, energy storing pneumohydraulic diaphragmis calibrated to fill said downstream microfluidic subcircuit to a mark.6. The microfluidic cartridge of claim 5, wherein each elastic, energystoring pneumohydraulic diaphragm is configured for filling a downstreamwettable microfluidic subcircuit equally to a mark, wherein each saidsubcircuit is configured for performing an assay in parallel, and eachsaid subcircuit is provided with a separate detection chamber.
 7. Themicrofluidic cartridge of claim 1, wherein said pneumohydraulicdiaphragm is a polyurethane diaphragm, a polyvinylidene chloridediaphragm, a polyolefin diaphragm, a polyester diaphragm, a polyethylenediaphragm, a polyethylene terephthalate diaphragm, a nylon diaphragm, ora laminated or co-extruded combination thereof.
 8. The microfluidiccartridge of claim l, wherein each said downstream vented channel isfluidily configured as an inlet to a microfluidic subcircuit, andwherein each said elastic, energy-storing pneumohydraulic diaphragm isconfigured for splitting a liquid volume equally between each saiddownstream microfluidic subcircuit.
 9. The microfluidic cartridge ofclaim 8, wherein said each microfluidic subcircuit comprises at leastone reaction chamber configured for mixing a liquid reagent, a dryreagent, or a combination thereof, with a liquid sample, and at leastone detection chamber configured for interfacing with a detection meansfor detecting a target analyte or analytes.
 10. The microfluidiccartridge of claim 1, wherein the microfluidic cartridge comprises anon-board reagent reservoir configured for dispensing a liquid volumeinto a microfluidic subcircuit of said hydraulic works; wherein saidreagent reservoir comprises: a) a duplexedly layered diaphragm sealedlyseparating a pneumatic chamber of said pneumatic works and a hydraulicchamber of said hydraulic works, said duplexedly layered diaphragmhaving a first side facing said pneumatic works and a second side facingsaid hydraulic works, a first layer forming said first side thereof, anda second layer forming said second side thereof, said first and secondlayers enclosing said liquid volume as a liquid center therebetween; b)a fluid outlet configured for receiving and conveying said liquid volumeto said downstream microfluidic subcircuit; and c) a sharp disposed insaid hydraulic chamber, said sharp configured for rupturing said secondlayer and for releasing said liquid volume into said hydraulic workswhen said duplexedly layered diaphragm is piercingly urged into contactwith the sharp by application of a pressure differential across saiddiaphragm.
 11. The microfluidic cartridge of claim 10, wherein saidfirst layer of said duplexedly layered diaphragm is rupture resistantand said second layer is rupture sensitive.
 12. The microfluidiccartridge of claim 11, wherein said first layer is a laminated polymerwith an outer nylon base layer configured to be puncture resistant andsaid second layer is a laminated polymer with an outer polyethyleneterephthalate member configured to be puncture susceptible.
 13. Themicrofluidic cartridge of claim 10, wherein said on-board reagentreservoir is configured for releasing serial liquid volumes by theaction of serial pulses of pneumatic pressure applied thereto.
 14. Themicrofluidic cartridge of claim 10, wherein said liquid volume comprisesa liquid reactant, a buffer, a rehydrating fluid, or a diluent, saidliquid volume for an assay step selected from rehydrating a dry reagentdisposed in a downstream chamber or channel, for rinsing a solid phase,for eluting a target analyte or analytes from a solid phase substrate,for making a dilution, for performing a chromatographic separation, foractuating or stopping a reaction, or for detecting said target analyteor analytes.
 15. The microfluidic cartridge of claim 1, wherein themicrofluidic cartridge comprises at least one microfluidic subcircuitwith a hydraulic chamber formed as a downstream reaction chamber with anupstream inlet and a downstream vent, said downstream reaction chambercontaining a dried reagent spot or spots and a pneumohydraulicdiaphragm, wherein said pneumohydraulic diaphragm is configured tooperate with a first position wherein the pneumohydraulic diaphragm isdistended against the floor of the downstream reaction chamber so as todisplace headspace air and form a protective temporary tent around andover the reagent spot or spots during wetout, and a second positionwherein the pneumohydraulic diaphragm is relaxedly positioned oraspirated against the roof of the downstream reaction chamber so as tofill the downstream reaction chamber with the liquid volume and uncoverand dissolve the reagent spot or spots at full strength without bubbleentrainment or reagent washout.
 16. The method of claim 15, wherein saiddried reagent spot or spots comprises a buffer, an enzyme, a co-enzyme,a co-factor, a polymerase, a primer, a molecular beacon, a probe, afluorophore, a dehydrogenase, an oxidase, a reactant, a chromogen, asubstrate, an antibody, an antigen, or a control.
 17. The microfluidiccartridge of claim 1, wherein the microfluidic cartridge is configuredfor performing PCR, and said microfluidic cartridge further comprises: afirst pneumohydraulic diaphragm overlying a first hydraulic chamber anda second pneumohydraulic diaphragm overlying a second hydraulic chamber,said first and second hydraulic chambers having a fluidicallyinterconnecting channel; a thermal interface for two-zone PCRthermocycling, with a first thermal interface of said first hydraulicchamber configured for apposing a first heating element and a secondthermal interface of said second hydraulic chamber configured forapposing a second heating element; and wherein said firstpneumohydraulic diaphragm is configured with a pneumatic means fordriving reciprocal fluid flow between said first and second hydraulicchambers during PCR amplification, and said interconnecting channel isconfigured to be operated at a tilt angle theta ranging from 10 to 35degrees so as to reduce interference from bubbles.
 18. The microfluidiccartridge of claim 17, wherein said second pneumohydraulic diaphragm isan elastomeric diaphragm and is configured for working passively by theurging of said first pneumohydraulic diaphragm.
 19. The microfluidiccartridge of claim 1, further comprising a detection chamber enclosed ona first opposite side by an optical window and on a second opposite sideby a thermo-optical window; wherein said detection chamber is configuredto be operated at a tilt angle theta ranging from 10 to 35 degrees so asto flush air and bubbles to a vented port superiorly disposed thereon.20. A kit comprising one or more microfluidic cartridges of claim 1 foruse as a consumable in a host instrument, said microfluidic cartridgecomprising on-board reagents for performing at least one assay for anucleic acid, a protein, an antigen, an antibody, a metabolite, or anenzyme.
 21. A kit comprising one or more microfluidic cartridges ofclaim 1 for use as a consumable in a host instrument, said microfluidiccartridge comprising on-board reagents for performing at least onenucleic acid assay, each cartridge packaged in a gas tight sealed pouch,with instructions for use, wherein the user need only place a biologicalliquid sample to be assayed in a sample inlet port and insert saidmicrofluidic cartridge into said host instrument, said cartridge havingall primers, enzyme-cofactors, salts, buffers, polymerase, and detectionchemistries for testing said liquid sample for a target nucleic acidassociated with a bacterium, a Rickettsia, a virus, a fungal agent, anantibiotic resistance gene, a gene associated with virulence ortoxigenicity, a molecular marker, a single-nucleotide polymorphism, aninsect gene, a bee disease agent gene, a plant gene, a plant diseaseagent, a molecular marker associated with a cell having a pathogenic orcarcinogenic condition, a mitochondrial nucleotide sequence, a plasmidsequence, a messenger RNA, a ribosomal RNA, or a panel of target nucleicacids.
 22. A method for wetout of a microfluidic cartridge comprisingreagent spots, while limiting bubble entrainment therein, the methodcomprising: a) pumping a liquid volume through an inlet and into aplurality of hydraulic chambers of the microfluidic card so that anelastic pneumohydraulic diaphragm overlying the liquid volume in eachsaid hydraulic chamber is distended, thereby isobarically pressurizingsaid liquid volume; b) pressurizing a second diaphragm in a plurality ofdownstream reaction chambers, each said downstream reaction chambercontaining a dried reagent spot, the second diaphragm forming aprotective temporary tent for sealing around and over the reagent spotand for displacing headspace air from the downstream reaction chamber;c) valvedly opening an outlet from each said hydraulic chamber, eachsaid outlet with fluidic connection to one of said plurality ofdownstream reaction chambers; d) wetting the downstream reaction chamberaround said temporary tent and displacing any residual air from thereaction chamber by allowing the distended elastic pneumohydraulicdiaphragm to relax, the liquid volume forming an advancing meniscus; e)optionally closing a valve downstream from the downstream reactionchamber; and f) lifting said temporary tent and conveying a remainingpart of the liquid volume into each reaction chamber, thereby dissolvingthe reagent spot at full strength without bubble entrainment or reagentwashout.
 23. The method of claim 22, wherein said dried reagent spot isa buffer, an enzyme, a co-enzyme, a co-factor, a polymerase, a primer, amolecular beacon, a probe, a fluorophore, a dehydrogenase, a reactant, achromogen, a substrate, an antibody, an antigen, or a control.
 24. Themicrofluidic cartridge of claim 1, wherein said pneumohydraulicdiaphragm comprises a metallized film layer.
 25. The microfluidiccartridge of claim 10, wherein said liquid volume is degassed and saidduplexedly layered diaphragm is gas impervious.
 26. The kit of claim 20,wherein the one or more microfluidic cartridges are provided in agas-tight package comprising inert atmosphere therein.
 27. The kit ofclaim 21, wherein: a) the bacterium is Acinetobacter baumannii,Actinobacillus equuli, Bacillus anthracis, Brucella melitensis, Brucellaabortus, Bordatella pertussis, Bordatella bronchioseptica, Burkholderiapseudomallei, Corynebacterium diptheriae, Coxiella burnetii, Eikenellacorrodens, Escherichia coli, Francisella tularensis, Francisellanovicida, Fusobacterium necrophorum, Haemophilus influenzae, Klebsiellaoxytoca, Klebsiella pneumoniae, Kingella denitrificans, Legionellapneumophila, Listeria monocytogenes, Moraxella catarrhalis,Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseriagonorrhoeae, Neisseria meningitides, Pasteurella multocida, Proteusvulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonasputrefaciens, Pseudomonas cepacia, Salmonella typhi, Shigelladysenteriae, Staphylococcus aureus, Streptococcus pyogenes,Streptococcus pneumoniae, Treponema pallidum, Yersinia pestis, or Vibriocholera; b) the Rickettsia is Chlamydia pneumoniae, Chlamydiatrachomatis, Rickettsia prowazekii, or Rickettsia typhi; c) the virus isMeasles virus, HIV virus, Hepatitis C virus, Hepatitis B virus, DengueVirus, Western Equine Encephalitis virus, Eastern Equine Encephalitisvirus, Venezuelan Equine Encephalitis virus, Enteroviruses, Influenzavirus, bird flu, Coronavirus, SARS Coronavirus, Polio virus, Adenovirus,Parainfluenza virus, Hanta virus, Rabies virus, Argentine HemorrhagicFever virus, Machupo virus, Sabia virus, Guanarito virus, Congo-CrimeanHemorrhagic Fever virus, Lassa Hemorrhagic Fever virus, Marburg virus,Ebola virus, Rift Valley Fever virus, Kyasanur Forest Disease virus,Omsk Hemorrhagic Fever, Yellow Fever virus, Smallpox virus, aretrovirus, Monkeypox virus, or foot and mouth disease virus; d) thefungal agent is Coccidiodes immitis, Candida albicans, Cryptococcusneoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Sporotrhixschenki, or Aspergillus fumigates; or e) the parasitic agent isPlasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Toxoplasma gondii, Plasmodium bergeri, Schistosoma mansoni,Schistosoma hematobium, Schistosoma japonicum, Entamoeba histolytica,Babesia, Toxoplasma gondii, Trypanosoma cruzi, Leishmania ssp,Trypanosoma brucei, Trichinella spiralis, Toxocara canis, Necatoramericanus, Trichuris trichura, Enterobius vermicularis, Dipylidiumcaninum, Entamoeba histolytica, Dracunculus medinensis, Wuchereriabancrofti, Brugia malai, Brugia timori, Strongyloides stercoralis,Ascaris lumbricoides, Onchocerca volvulus, Naegleria fowleri, Clonorchissinensis, Cryptosporidium parvum, or Leishmania spp.
 28. A microfluidiccartridge comprising a plastic, thermally insulative cartridge bodyenclosing: i) a hydraulic works comprising an upstream sample inlet, oneor more on-board liquid or dry reagents for an assay, and a wettabledownstream microfluidic subcircuit comprising a channel and chamberfluidly connected to said upstream inlet and vented at a downstreamvent; and ii) a pneumatic works comprising an inlet or inlets forreceiving pneumatic pressure and a pneumatic subcircuit comprising achannel and chamber for conveying said pneumatic pressure therefrom; andiii) an on-board reagent reservoir configured for dispensing a liquidvolume into a microfluidic subcircuit of said hydraulic works; whereinsaid reagent reservoir comprises: a) a duplexedly layered diaphragmsealedly separating a pneumatic chamber of said pneumatic works and ahydraulic chamber of said hydraulic works, said duplexedly layereddiaphragm having a first side facing said pneumatic works and a secondside facing said hydraulic works, a first layer forming said first sidethereof, and a second layer forming said second side thereof, said firstand second layers enclosing said liquid volume as a liquid centertherebetween; b) a fluid outlet configured for receiving and conveyingsaid liquid volume to said downstream microfluidic subcircuit; and c) asharp disposed in said hydraulic chamber, said sharp configured forrupturing said second layer and for releasing said liquid volume intosaid hydraulic works when said duplexedly layered diaphragm ispiercingly urged into contact with the sharp by application of apressure differential across said diaphragm, wherein said hydraulicchamber and duplexedly layered diaphragm are configured for preventingor reducing bubble entrainment or reagent washout during wetout, fill,pumping or rehydration steps of an assay, wherein said hydraulic worksis configured for operation when mounted at a tilt angle theta rangingfrom 10 to 35 degrees relative to the ground plane on a tilted stage ofa host instrument and at least one hydraulic chamber is configured withan outlet and intercommunicating channel positioned superiorly relativeto said hydraulic chamber for venting a gas or discharging a bubble fromsaid hydraulic chamber.
 29. The microfluidic cartridge of claim 28,wherein said first layer of said duplexedly layered diaphragm is ruptureresistant and said second layer is rupture sensitive.
 30. Themicrofluidic cartridge of claim 29, wherein said first layer is alaminated polymer with an outer nylon base layer configured to bepuncture resistant and said second layer is a laminated polymer with anouter polyethylene terephthalate member configured to be puncturesusceptible.
 31. The microfluidic cartridge of claim 28, wherein saidon-board reagent reservoir is configured for releasing serial liquidvolumes by the action of serial pulses of pneumatic pressure appliedthereto.
 32. The microfluidic cartridge of claim 28, wherein said liquidvolume comprises a liquid reactant, a buffer, a rehydrating fluid, or adiluent, said liquid volume for an assay step selected from rehydratinga dry reagent disposed in a downstream chamber or channel, for rinsing asolid phase, for eluting a target analyte or analytes from a solid phasesubstrate, for making a dilution, for performing a chromatographicseparation, for actuating or stopping a reaction, or for detecting saidtarget analyte or analytes.
 33. The microfluidic cartridge of claim 28,wherein said liquid volume is degassed and said duplexedly layereddiaphragm is gas impervious.
 34. The microfluidic cartridge of claim 28,wherein said hydraulic works comprises a plurality of wettabledownstream microfluidic subcircuits with channels and chambers fluidlyconnected to said upstream inlet and vented at one or more downstreamvents, wherein said plurality of wettable downstream microfluidicsubcircuits are each configured for performing an assay in parallel, andeach said wettable downstream microfluidic subcircuit is provided with aseparate detection chamber.
 35. The microfluidic cartridge of claim 28,further comprising an energy-storing pneumohydraulic diaphragm, andwherein the microfluidic cartridge is configured such that a hydraulicpressure and liquid volume of said elastic, energy storingpneumohydraulic diaphragm in a distended state is passively converted toa work of advancing a meniscus in a wettable downstream microfluidicsubcircuit fluidly connected thereto by opening a downstream valve,thereby displacing any gas to said downstream vent.
 36. The microfluidiccartridge of claim 35, wherein said liquid volume and hydraulic pressureof said elastic, energy storing pneumohydraulic diaphragm is calibratedto fill said downstream microfluidic subcircuit to a mark.
 37. Themicrofluidic cartridge of claim 36, wherein each comprising a pluralityof elastic, energy storing pneumohydraulic diaphragm, and each elastic,energy storing pneumohydraulic diaphragm is configured for filling adownstream wettable microfluidic subcircuit equally to a mark, whereineach said subcircuit is configured for performing an assay in parallel,and each said subcircuit is provided with a separate detection chamber.38. The microfluidic cartridge of claim 28, wherein the microfluidiccartridge further comprises: a) a staging manifold having a plurality ofchambers, wherein each said chamber of said plurality of chambers isseparated into a hydraulic chamber and a pneumatic chamber by anelastic, energy-storing pneumohydraulic diaphragm sealedly mountedtherebetween, such that a liquid volume admitted through an inlet intoeach said hydraulic chamber in series or in parallel distends each saidenergy-storing pneumohydraulic diaphragm according to an isobaricpressure proportionate to the displacement volume thereof, wherein saidinlet is valvedly closeable for equilibrating said hydraulic pressurethroughout said staging manifold after filling is complete; and; b) aplurality of vented downstream channels in parallel, wherein one saidchannel of said plurality of channels is in fluidic communication withat least one of said hydraulic chambers of said staging manifold, eachsaid vented downstream channel having a valve configured for closingduring filling and pressurization and for opening during draining anddepressurization, whereby said hydraulic pressure of said elastic,pneumohydraulic diaphragm in a distended state is passively converted tothe work of advancing a meniscus during initial wetout of said pluralityof vented downstream channels in parallel.
 39. The microfluidiccartridge of claim 38, wherein each said downstream vented channel isfluidily configured as an inlet to a microfluidic subcircuit, andwherein each said elastic, energy-storing pneumohydraulic diaphragm isconfigured for splitting a liquid volume equally between each saiddownstream microfluidic subcircuit.
 40. The microfluidic cartridge ofclaim 39, wherein said each microfluidic subcircuit comprises at leastone reaction chamber configured for mixing a liquid reagent, a dryreagent, or a combination thereof, with a liquid sample, and at leastone detection chamber configured for interfacing with a detection meansfor detecting a target analyte or analytes.
 41. The microfluidiccartridge of claim 28, wherein said pneumatic chamber is vented toatmosphere.
 42. The microfluidic cartridge of claim 28, wherein themicrofluidic cartridge further comprises at least one microfluidicsubcircuit with a hydraulic chamber formed as a downstream reactionchamber with an upstream inlet and a downstream vent, said downstreamreaction chamber containing a dried reagent spot or spots and apneumohydraulic diaphragm, wherein said pneumohydraulic diaphragm isconfigured to operate with a first position wherein the pneumohydraulicdiaphragm is distended against the floor of the downstream reactionchamber so as to displace headspace air and form a protective temporarytent around and over the reagent spot or spots during wetout, and asecond position wherein the pneumohydraulic diaphragm is relaxedlypositioned or aspirated against the roof of the downstream reactionchamber so as to fill the downstream reaction chamber with the liquidvolume and uncover and dissolve the reagent spot or spots at fullstrength without bubble entrainment or reagent washout.
 43. The methodof claim 42, wherein said dried reagent spot or spots comprises abuffer, an enzyme, a co-enzyme, a co-factor, a polymerase, a primer, amolecular beacon, a probe, a fluorophore, a dehydrogenase, an oxidase, areactant, a chromogen, a substrate, an antibody, an antigen, or acontrol.
 44. The microfluidic cartridge of claim 28, wherein themicrofluidic cartridge is configured for performing PCR, and saidmicrofluidic cartridge further comprises: a first pneumohydraulicdiaphragm overlying a first hydraulic chamber and a secondpneumohydraulic diaphragm overlying a second hydraulic chamber, saidfirst and second hydraulic chambers having a fluidically interconnectingchannel; a thermal interface for two-zone PCR thermocycling, with afirst thermal interface of said first hydraulic chamber configured forapposing a first heating element and a second thermal interface of saidsecond hydraulic chamber configured for apposing a second heatingelement; and wherein said first pneumohydraulic diaphragm is configuredwith a pneumatic means for driving reciprocal fluid flow between saidfirst and second hydraulic chambers during PCR amplification, and saidinterconnecting channel is configured to be operated at a tilt angletheta ranging from 10 to 35 degrees so as to reduce interference frombubbles.
 45. The microfluidic cartridge of claim 44, wherein said secondpneumohydraulic diaphragm is an elastomeric diaphragm and is configuredfor working passively by the urging of said first pneumohydraulicdiaphragm.
 46. The microfluidic cartridge of claim 28, furthercomprising a detection chamber enclosed on a first opposite side by anoptical window and on a second opposite side by a thermo-optical window;wherein said detection chamber is configured to be operated at a tiltangle theta ranging from 10 to 35 degrees so as to flush air and bubblesto a vented port superiorly disposed thereon.
 47. A kit comprising oneor more microfluidic cartridges of claim 28 for use as a consumable in ahost instrument, said microfluidic cartridge comprising on-boardreagents for performing at least one assay for a nucleic acid, aprotein, an antigen, an antibody, a metabolite, or an enzyme.
 48. Thekit of claim 47, wherein the one or more microfluidic cartridges areprovided in a gas-tight package comprising inert atmosphere therein. 49.A kit comprising one or more microfluidic cartridges of claim 28 for useas a consumable in a host instrument, said microfluidic cartridgecomprising on-board reagents for performing at least one nucleic acidassay, each cartridge packaged in a gas tight sealed pouch, withinstructions for use, wherein the user need only place a biologicalliquid sample to be assayed in a sample inlet port and insert saidmicrofluidic cartridge into said host instrument, said cartridge havingall primers, enzyme-cofactors, salts, buffers, polymerase, and detectionchemistries for testing said liquid sample for a target nucleic acidassociated with a bacterium, a Rickettsia, a virus, a fungal agent, anantibiotic resistance gene, a gene associated with virulence ortoxigenicity, a molecular marker, a single-nucleotide polymorphism, aninsect gene, a bee disease agent gene, a plant gene, a plant diseaseagent, a molecular marker associated with a cell having a pathogenic orcarcinogenic condition, a mitochondrial nucleotide sequence, a plasmidsequence, a messenger RNA, a ribosomal RNA, or a panel of target nucleicacids.
 50. The kit of claim 49, wherein: a) the bacterium isAcinetobacter baumannii, Actinobacillus equuli, Bacillus anthracis,Brucella melitensis, Brucella abortus, Bordatella pertussis, Bordatellabronchioseptica, Burkholderia pseudomallei, Corynebacterium diptheriae,Coxiella burnetii, Eikenella corrodens, Escherichia coli, Francisellatularensis, Francisella novicida, Fusobacterium necrophorum, Haemophilusinfluenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Kingelladenitrificans, Legionella pneumophila, Listeria monocytogenes, Moraxellacatarrhalis, Mycobacterium tuberculosis, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitides, Pasteurella multocida,Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa, Pseudomonasputrefaciens, Pseudomonas cepacia, Salmonella typhi, Shigelladysenteriae, Staphylococcus aureus, Streptococcus pyogenes,Streptococcus pneumoniae, Treponema pallidum, Yersinia pestis, or Vibriocholera; b) the Rickettsia is Chlamydia pneumoniae, Chlamydiatrachomatis, Rickettsia prowazekii, or Rickettsia typhi; c) the virus isMeasles virus, HIV virus, Hepatitis C virus, Hepatitis B virus, DengueVirus, Western Equine Encephalitis virus, Eastern Equine Encephalitisvirus, Venezuelan Equine Encephalitis virus, Enteroviruses, Influenzavirus, bird flu, Coronavirus, SARS Coronavirus, Polio virus, Adenovirus,Parainfluenza virus, Hanta virus, Rabies virus, Argentine HemorrhagicFever virus, Machupo virus, Sabia virus, Guanarito virus, Congo-CrimeanHemorrhagic Fever virus, Lassa Hemorrhagic Fever virus, Marburg virus,Ebola virus, Rift Valley Fever virus, Kyasanur Forest Disease virus,Omsk Hemorrhagic Fever, Yellow Fever virus, Smallpox virus, aretrovirus, Monkeypox virus, or foot and mouth disease virus; d) thefungal agent is Coccidiodes immitis, Candida albicans, Cryptococcusneoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Sporotrhixschenki, or Aspergillus fumigates; or e) the parasitic agent isPlasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Toxoplasma gondii, Plasmodium bergeri, Schistosoma mansoni,Schistosoma hematobium, Schistosoma japonicum, Entamoeba histolytica,Babesia, Toxoplasma gondii, Trypanosoma cruzi, Leishmania ssp,Trypanosoma brucei, Trichinella spiralis, Toxocara canis, Necatoramericanus, Trichuris trichura, Enterobius vermicularis, Dipylidiumcaninum, Entamoeba histolytica, Dracunculus medinensis, Wuchereriabancrofti, Brugia malai, Brugia timori, Strongyloides stercoralis,Ascaris lumbricoides, Onchocerca volvulus, Naegleria fowleri, Clonorchissinensis, Cryptosporidium parvum, or Leishmania spp.